Methods for aiding in diagnosing and evaluating a mild traumatic brain injury in a human subject using cardiac troponin i and early biomarkers

ABSTRACT

Disclosed herein are methods that aid in the diagnosis and evaluation of a human subject that has sustained or may have sustained an injury to the head, such as mild or a moderate, severe, or moderate to severe traumatic brain injury (TBI), by detecting levels of cardiac troponin I (cTnI) and one or more early biomarkers which are not cTnI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in biological samples taken from a human subject at time points within about 24 hours of injury after the subject has sustained or may have sustained the injury to the head.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. application Ser. No.15/993,588 filed May 30, 2018, which claims priority to U.S. ApplicationNo. 62/512,688 filed on May 30, 2017, U.S. Application No. 62/512,710filed on May 30, 2017 and U.S. Application No. 62/528,214 filed on Jul.3, 2017, the contents of each of which are herein incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 29, 2020, isnamed 36103-303_ST25.txt and is 8,523 bytes in size.

TECHNICAL FIELD

The present disclosure relates to methods of aiding in diagnosing andevaluating a human subject that has sustained or may have sustained an(or has an actual or suspected) injury to the head, such as mildtraumatic brain injury (TBI), by detecting levels of cardiac troponin I(cTnI) and one or more early biomarkers which are not cTnI, such asubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillaryacidic protein (GFAP), or a combination thereof, in biological samplestaken from a human subject at time points after an actual injury orsuspected injury to the head.

BACKGROUND

More than 5 million mild traumatic brain injuries (TBIs) occur each yearin the United States alone. Currently, there is no simple, objective,accurate measurement available to help in patient assessment. In fact,much of TBI evaluation and diagnosis is based on subjective data.Unfortunately, objective measurements such as head CT and Glasgow ComaScore (GCS) are not very comprehensive or sensitive in evaluating mildTBI. Moreover, head CT is unrevealing for the vast majority of the timefor mild TBI, is expensive, and exposes the patient to unnecessaryradiation. Additionally, a negative head CT does not mean the patienthas been cleared from having a concussion; rather it just means certaininterventions, such as surgery, are not warranted. Clinicians andpatients need objective, reliable information to accurately evaluatethis condition to promote appropriate triage and recovery. To date,limited data have been available for the use of cardiac troponin I inthe acute care setting or hyperacute care setting (very early acute timepoints after injury) to aid in patient evaluation and management.

Mild TBI or concussion is much harder to objectively detect and presentsan everyday challenge in emergency care units globally. Concussionusually causes no gross pathology, such as hemorrhage, and noabnormalities on conventional computed tomography scans of the brain,but rather rapid-onset neuronal dysfunction that resolves in aspontaneous manner over a few days to a few weeks. There is an unmetneed for mild TBI victims on scene, in emergency rooms and clinics, inthe sports area and in military activity (e.g., combat).

Current algorithms for assessment of the severity of brain injuryinclude Glasgow Coma Scale score and other measures. These measures mayat times be adequate for relating acute severity but are insufficientlysensitive for subtle pathology which can result in persistent deficit.GCS and other measures also do not enable differentiation among types ofinjury and may not be adequate. Thus patients grouped into a single GCSlevel entering a clinical trial may have vastly heterogeneous severityand type of injury. Because outcomes also vary accordingly,inappropriate classification undermines the integrity of a clinicaltrial. Improved classification of injury will enable more precisedelineation of disease severity and type for TBI patients in clinicaltrials.

Additionally, current brain injury trials rely on outcome measures suchas Glasgow Outcome Scale Extended, which capture global phenomena butfail to assess for subtle differences in outcome. Thus 30 consecutivetrials for brain injury therapeutics have failed. Sensitive outcomemeasures are needed to determine how well patients have recovered frombrain injury in order to test therapeutics and prophylactics.

Traumatic brain injury (TBI) patients are at least three times morelikely to die from cardiovascular causes than the general population.Cardiac injury from TBI is also associated with neurogenic pulmonaryedema. The phenomenon of cardiac injury in neurologic conditions hasbeen described in patients with spontaneous subarachnoid hemorrhage andis believed to result from a fulminant surge in catecholamine levels.However, the mechanisms underlining excess cardiovascular mortality inTBI have been poorly studied and therefore are not well understood.Consequently, it is unclear whether: the onset of cardiac injury occursin the acute or chronic phase of TBI; there are particular sub-types ofTBI that are preferentially affected by cardiac injury; and what thebiological triggers of cardiac injury in TBI are. A number ofretrospective studies have investigated myocardial injury in the acutephase of TBI. Using conventional cardiac troponin assays, these studieshave reported cardiac injury (as determined by elevated troponin levels)within 24 hours of injury in 30% of severe TBI patient. Cardiac injuryin TBI is associated with injury severity and age. TBI patients withcardiac injury have a higher risk of in-patient mortality than thosewithout cardiac injury. However, these findings are subject to spectrumbias since they are derived from retrospective studies and troponinmeasurements were performed at the discretion of clinicians (they arerarely done in the routine care of TBI patients). Furthermore, theassociation between cardiac injury and neurologic outcome in TBI has notbeen studied. Additionally, the role of cardiac injury in mild andmoderate TBI has not been studied.

SUMMARY

The present disclosure is directed to a method for aiding in thediagnosis and evaluation of mild traumatic brain injury in a humansubject. The method comprises: a) performing an assay on a sampleobtained from the subject within about 24 hours after a an actual orsuspected injury to the head to measure or detect a level of cardiactroponin I (cTnI) and a level of an early biomarker, wherein the sampleis a biological sample and early biomarker comprises ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof; and b) determining whether the subjecthas sustained a mild or moderate, severe, or a moderate to severetraumatic brain injury (TBI), wherein the subject is determined ashaving (1) a moderate, severe, or a moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI and the level of the early biomarker in the sample ishigher than a reference level of the early biomarker; or (2) a mildtraumatic brain injury when the level of cTnI in the sample is lowerthan a reference level of cTnI and/or the level of the early biomarkerin the sample is lower than a reference level of the early biomarker.

In some embodiments of the above method, the subject is diagnosed ordetermined to have sustained a mild traumatic brain injury. In otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a moderate traumatic brain injury. In yet otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a severe traumatic brain injury. In yet otherembodiments, the subject is diagnosed or determined to have sustained amoderate to severe traumatic brain injury.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments in the above method, the subject is suspected ashaving a moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

In some embodiments in the above method, the reference levels of cTnIand early biomarker are correlated with (corresponds to) a mildtraumatic brain injury. In some embodiments in the above method, thereference levels of cTnI and early biomarker are correlated with(corresponds to) a moderate traumatic brain injury. In other embodimentsof the above method, the reference levels of cTnI and early biomarkercorrelated with (corresponds to) a severe traumatic brain injury. Insome embodiments in the above method, the reference levels of cTnI andearly biomarkers are correlated with (correspond to) a moderate tosevere traumatic brain injury.

In some embodiments, the subject may be suspected as having mild TBIbased on the Glasgow Coma Scale score. In other aspects, the subject maybe suspected of having a moderate TBI based on the Glasgow Coma Scalescore. In other aspects, the subject may be suspected of having a severeTBI based on the Glasgow Coma Scale Score. In other aspects, the subjectmay be suspect as having a moderate to severe TBI based on the GlasgowComa scale score. In other aspects, the reference level of GFAP or thereference level correlates with or correspond to a Glasgow Coma Scalescore of 13-15 (a mild TBI). In other aspects, the reference levelcorrelates or correspond to a Glasgow Coma Scale score of 3-8 (a severeTBI). In other aspects, the reference level correlates or correspond toa Glasgow Coma Scale score of 9-13 (a moderate TBI). In other aspects,the reference level correlates with or correspond to a Glasgow ComaScale score of 3-12 (a moderate to severe TBI).

In some embodiments of the above method, the reference level for cTnI isabout 5 pg/mL. In some embodiments of the above method, the referencelevel for cTnI is about 10 pg/mL. In some embodiments of the abovemethod, the reference level for cTnI is about 15 pg/mL. In someembodiments of the above method, the reference level for cTnI is about20 pg/mL. In some embodiments of the above method, the reference levelfor cTnI is about 35 pg/mL. In some embodiments of the above method, thereference level for cTnI is about 50 pg/mL.

In some embodiments of the above method, the reference level for UCH-L1is about 400 pg/mL. In some embodiments of the above method, thereference level for UCH-L1 is about 500 pg/mL. In some embodiments ofthe above method, the reference level for UCH-L1 is about 550 pg/mL.

In some embodiments of the above method, the reference level for GFAP isabout 70 pg/mL. In some embodiments of the above method, the referencelevel for GFAP is about 100 pg/mL. In some embodiments of the abovemethod, the reference level for GFAP is about 150 pg/mL.

In some embodiments of the above method, the reference level isdetermined by an assay having a sensitivity of between at least about85% to 100% and a specificity of between at least about 30% to 100%. Insome embodiments of the above method, the reference level is determinedby an assay having a sensitivity of at least about 87.5% and aspecificity of at least about 31%. In some embodiments of the aboveassay, the reference level is between at least about 1 pg/mL to about100 pg/mL. In some embodiments of the above assay, the reference levelis between at least about 1 pg/mL to about 500 pg/mL. In someembodiments of the above method, the reference level is between at leastabout 1 pg/mL to about 1000 pg/mL.

In some embodiments in the above method, the sample is taken withinabout 30 minutes, within about 1 hour, within about 2 hours, withinabout 3 hours, within about 4 hours, within about 5 hours, within about6 hours, within about 7 hours, within about 8 hours, within about 9hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of the actual or suspected injury to the head. Specifically, insome embodiments of the above method, the sample is taken within about30 minutes of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 1 hourof the actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 2 hours of the actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 3 hours of the actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 4 hours of the actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 5 hours of the actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 6 hours of the actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about7 hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 8hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 9hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 10hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 11hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 12hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 13hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 14hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 15hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 16hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 17hours of the actual or suspected injury to the head the actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 18 hours of the actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 19 hours of the actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 20 hours of the actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about21 hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 22hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 23hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 24hours of the actual or suspected injury to the head.

In some embodiments, the above method further comprises treating thesubject assessed as having a moderate, severe or moderate to severetraumatic brain injury with a traumatic brain injury treatment. In someembodiments, the above method further comprises monitoring the subjectassessed as having a moderate, severe, or moderate to severe traumaticbrain injury before treatment with a traumatic brain injury treatment.In some embodiments, the above method further comprises monitoring thesubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury after treatment with a traumatic brain injurytreatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In another embodiment, the present disclosure is directed to a method ofaiding in the determination of whether to perform a head computerizedtomography (CT) scan on a human subject that has sustained or may havesustained an (or has an actual or suspected) injury to the head. Themethod comprises: a) performing an assay on a sample obtained from thesubject within about 24 hours after an actual or suspected injury to thehead to measure or detect a level of cTnI and a level of an earlybiomarker in the sample, wherein the sample is a biological sample andearly biomarker comprises UCH-L1, GFAP, or a combination thereof; and b)performing a CT scan on the subject when the level of cTnI in the sampleis higher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker and not performing a CT scan on the subject when the level ofcTnI in the sample is lower than a reference level of cTnI and/or thelevel of the early biomarker in the sample is lower than a referencelevel of the early biomarker.

In some embodiments in the above method, the sample is taken withinabout 30 minutes, within about 1 hour, within about 2 hours, withinabout 3 hours, within about 4 hours, within about 5 hours, within about6 hours, within about 7 hours, within about 8 hours, within about 9hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours, or within about 24hours of the actual or suspected injury to the head. Specifically, insome embodiments of the above method, the sample is taken within about30 minutes of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 1 hourof the actual or suspected injury to the head. In other embodiments ofthe above method, the sample is taken within about 2 hours of the actualor suspected injury to the head. In other embodiments of the abovemethod, the sample is taken within about 3 hours of the actual orsuspected injury to the head. In other embodiments of the above method,the sample is taken within about 4 hours of the actual or suspectedinjury to the head. In other embodiments of the above method, the sampleis taken within about 5 hours of the actual or suspected injury to thehead. In other embodiments of the above method, the sample is takenwithin about 6 hours of the actual or suspected injury to the head. Inother embodiments of the above method, the sample is taken within about7 hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 8hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 9hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 10hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 11hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 12hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 13hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 14hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 15hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 16hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 17hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 18hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 19hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 20hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 21hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 22hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 23hours of the actual or suspected injury to the head. In otherembodiments of the above method, the sample is taken within about 24hours of the actual or suspected injury to the head.

In some embodiments of the above method, a CT scan is performed on thesubject. In other embodiments of the above method, a CT scan is notperformed on the subject.

In some embodiments of the above-described method, the subject hasreceived a CT scan before or after the assay is performed, and whereinthe subject is suspected as having a TBI based on the CT scan result. Insome embodiments, the subject may be suspected of having a traumaticbrain injury based on a CT scan that already was performed. For example,depending upon a subject's medical condition (such as, if the patient isunconscious), a CT scan may be conducted shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a CT scan maybe performed prior to the assay being performed to confirm and determinewhether the subject has a mild or moderate, severe, or a moderate tosevere TBI. After the assay is performed, one or more subsequent CTscans can be performed based on the results of the assay as part of thephysician's (or other medical personnel's) management of the TBI (suchas, for example, to determine whether surgical and/or pharmacologicalintervention may be required). In other embodiments, the subject may nothave received a CT scan before the assay is performed.

In some embodiments in the above method, the subject is suspected ofhaving a traumatic brain injury based on the CT scan. In someembodiments, the subject is diagnosed as having a traumatic brain injurybased on the CT scan. In other embodiments, the subject is diagnosed asnot having a traumatic brain injury based on the CT scan.

In some embodiments in the above method, the reference levels of cTnIand of the early biomarker are correlated with (corresponds to) apositive head computed tomography.

In some embodiments in the above method, the reference levels of cTnIand of the early biomarker correlated with (corresponds to) controlsubjects that have not sustained a head injury.

In some embodiments of the above method, the reference level for cTnI isabout 5 pg/mL. In some embodiments of the above method, the referencelevel for cTnI is about 10 pg/mL. In some embodiments of the abovemethod, the reference level for cTnI is about 15 pg/mL. In someembodiments of the above method, the reference level for cTnI is about20 pg/mL. In some embodiments of the above method, the reference levelfor cTnI is about 35 pg/mL. In some embodiments of the above method, thereference level for cTnI is about 50 pg/mL.

In some embodiments of the above method, the reference level for UCH-L1is about 400 pg/mL. In some embodiments of the above method, thereference level for UCH-L1 is about 500 pg/mL. In some embodiments ofthe above method, the reference level for UCH-L1 is about 550 pg/mL.

In some embodiments of the above method, the reference level for GFAP isabout 50 pg/mL. In some embodiments of the above method, the referencelevel for GFAP is about 100 pg/mL. In some embodiments of the abovemethod, the reference level for GFAP is about 150 pg/mL.

In embodiments of the above method, the reference level is determined byan assay having a sensitivity of between at least about 65% to 100% anda specificity of between at least about 29% to 100%. In embodiments ofthe above method, the reference level determined by an assay having asensitivity of at least about 85% and a specificity of at least about33%. In embodiments of the above method, the reference level isdetermined between at least about 1 pg/mL to about 100 pg/mL. Inembodiments of the above method, the reference level is between at leastabout 1 pg/mL to about 500 pg/mL. In embodiments of the above method,the reference level is between at least about 1 pg/mL to about 1000pg/mL.

In another embodiment, the present disclosure is directed to a method ofaiding in the diagnosis and evaluation of a human subject that hassustained or may have sustained an (or has an actual or suspected)injury to the head. The method comprises: a) performing an assay on asample obtained from the subject within about 2 hours after an actual orsuspected injury to the head to measure or detect a level of cTnI and alevel of an early biomarker, wherein the sample is a biological sampleand early biomarker comprises UCH-L1, GFAP, or a combination thereof;and b) determining whether the subject has sustained a mild or amoderate, severe, or moderate to severe traumatic brain injury (TBI),wherein the subject is determined as having (1) a moderate, severe, or amoderate to severe traumatic brain injury when the level of cTnI in thesample is higher than a reference level of cTnI and the level of theearly biomarker in the sample is higher than a reference level of theearly biomarker or (2) a mild traumatic brain injury when the level ofcTnI in the sample is lower than a reference level of cTnI and/or thelevel of the early biomarker in the sample is lower than a referencelevel of the early biomarker.

In some embodiments of the above method, the subject is diagnosed ordetermined to have sustained a mild traumatic brain injury. In otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a moderate traumatic brain injury. In yet otherembodiments of the above method, the subject is diagnosed or determinedto have sustained a severe traumatic brain injury. In yet otherembodiments, the subject is diagnosed or determined to have sustained amoderate to severe traumatic brain injury.

In some embodiments in the above method, the subject has received aGlasgow Coma Scale score before or after the assay is performed. In someembodiments, the subject may be suspected of having a traumatic braininjury based on a Glasgow Coma Scale score that was previouslyperformed. For example, depending upon a subject's medical condition, aGlasgow Coma Scale score may be assessed shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a GlasgowComa Scale score may be provided prior to the assay being performed toconfirm and determine whether the subject has a mild or moderate,severe, or moderate to severe TBI. After the assay is performed, one ormore subsequent Glasgow Coma Scale scores can be performed based on theresults of the assay as part of the physician's (or other medicalpersonnel's) management of the TBI (such as, for example, to determinewhether surgical and/or pharmacological intervention may be required).In other embodiments, the subject may not have received a Glasgow ComaScale score before the assay is performed.

In some embodiments in the above method, the subject is suspected ashaving a moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

In some embodiments in the above method, the reference levels of cTnIand early biomarker are correlated with (corresponds to) a mildtraumatic brain injury. In some embodiments in the above method, thereference levels of cTnI and early biomarker are correlated with(corresponds to) a moderate traumatic brain injury. In other embodimentsof the above method, the reference levels of cTnI and early biomarkercorrelated with (corresponds to) a severe traumatic brain injury. Insome embodiments in the above method, the reference levels of cTnI andearly biomarkers are correlated with (correspond to) a moderate tosevere traumatic brain injury.

In some embodiments, the subject may be suspected as having mild TBIbased on the Glasgow Coma Scale score. In other aspects, the subject maybe suspected of having a moderate TBI based on the Glasgow Coma Scalescore. In other aspects, the subject may be suspected of having a severeTBI based on the Glasgow Coma Scale Score. In other aspects, the subjectmay be suspect as having a moderate to severe TBI based on the GlasgowComa scale score. In other aspects, the reference level of GFAP or thereference level correlates with or correspond to a Glasgow Coma Scalescore of 13-15 (a mild TBI). In other aspects, the reference levelcorrelates or correspond to a Glasgow Coma Scale score of 3-8 (a severeTBI). In other aspects, the reference level correlates or correspond toa Glasgow Coma Scale score of 9-13 (a moderate TBI). In other aspects,the reference level correlates with or correspond to a Glasgow ComaScale score of 3-12 (a moderate to severe TBI).

In some embodiments, the above method further comprises treating thesubject assessed as having a moderate, severe or moderate to severetraumatic brain injury with a traumatic brain injury treatment. In someembodiments, the above method further comprises monitoring the subjectassessed as having a moderate, severe, or moderate to severe traumaticbrain injury before treatment with a traumatic brain injury treatment.In some embodiments, the above method further comprises monitoring thesubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury after treatment with a traumatic brain injurytreatment.

In some embodiments, the above method further comprises monitoring thesubject assessed as having mild traumatic brain injury. In someembodiments, the above method further comprises treating the subjectassessed as having a mild traumatic brain injury with a traumatic braininjury treatment. In some embodiments, the above method comprisesmonitoring the subject assessed as having a mild traumatic brain injurybefore treating with a traumatic brain injury treatment. In otherembodiments, the above method comprises monitoring the subject assessedas having a mild traumatic brain injury after treatment with a traumaticbrain injury treatment.

In yet another embodiment, the present disclosure is directed to amethod of aiding in the determination of whether to perform a headcomputerized tomography (CT) scan on a human subject that has sustainedor may have sustained an (or has an actual or suspected) injury to thehead. The method comprises: a) performing an assay on a sample obtainedfrom the subject within about 2 hours after an actual or suspectedinjury to the head to measure or detect a level of cTnI and a level ofan early biomarker in the sample, wherein the sample is a biologicalsample and early biomarker comprises UCH-L1, GFAP, or a combinationthereof; and b) performing a CT scan on the subject when the level ofcTnI in the sample is higher than a reference level of cTnI and thelevel of the early biomarker in the sample is higher than a referencelevel of the early biomarker and not performing a CT scan on the subjectwhen the level of cTnI in the sample is lower than a reference level ofcTnI and/or the level of the early biomarker in the sample is lower thana reference level of the early biomarker.

In some embodiments of the above method, a CT scan is performed on thesubject. In other embodiments of the above method, a CT scan is notperformed on the subject.

In some embodiments of the above-described method, the subject hasreceived a CT scan before or after the assay is performed, and whereinthe subject is suspected as having a TBI based on the CT scan result. Insome embodiments, the subject may be suspected of having a traumaticbrain injury based on a CT scan that already was performed. For example,depending upon a subject's medical condition (such as, if the patient isunconscious), a CT scan may be conducted shortly after the subjectarrives at an emergency room, trauma center, or other site in order toassess and/or evaluate whether the subject has a TBI. Such a CT scan maybe performed prior to the assay being performed to confirm and determinewhether the subject has a mild or a moderate, severe, or a moderate tosevere TBI. After the assay is performed, one or more subsequent CTscans can be performed based on the results of the assay as part of thephysician's (or other medical personnel's) management of the TBI (suchas, for example, to determine whether surgical and/or pharmacologicalintervention may be required). In other embodiments, the subject may nothave received a CT scan before the assay is performed.

In some embodiments in the above method, the subject is suspected ofhaving a traumatic brain injury based on the CT scan. In someembodiments, the subject is diagnosed as having a traumatic brain injurybased on the CT scan. In other embodiments, the subject is diagnosed asnot having a traumatic brain injury based on the CT scan.

In some embodiments in the above method, the reference levels of cTnIand of the early biomarker are correlated with (corresponds to) apositive head computed tomography.

In some embodiments in the above method, the reference levels of cTnIand of the early biomarker correlated with (corresponds to) controlsubjects that have not sustained a head injury.

In another embodiment, the present disclosure relates to a method oftreating a mild, moderate, severe, or moderate to severe traumatic braininjury in a human subject, the method comprising: a) performing an assayon a sample obtained from the subject within about 24 hours after anactual or suspected injury to the head to measure or detect a level ofcTnI and a level of an early biomarker, wherein the sample is abiological sample and the early biomarker comprises UCH-L1, GFAP, or acombination thereof; b) determining whether the subject has sustained amild or a moderate, severe, or moderate to severe traumatic brain injury(TBI), wherein the subject is determined as having (1) a moderate,severe, or a moderate to severe traumatic brain injury when the level ofcTnI in the sample is higher than a reference level of cTnI and thelevel of the early biomarker in the sample is higher than a referencelevel of the early biomarker or (2) a mild traumatic brain injury whenthe level of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker; and c) treating the subjectassessed as having a mild, moderate, severe, or moderate to severetraumatic brain injury with a traumatic brain injury treatment.

In some embodiments of the above method, the traumatic brain injurytreatment for a subject suffering from a mild TBI can involve having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. In other embodiments of the above method,the traumatic brain injury treatment for a subject suffering from amoderate, severe, or moderate to severe TBI, the treatment involves theadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt or providing of oneor more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof. Optionally, such methods may alsoinvolve providing one or more cardioprotective therapies. Suchcardioprotective therapies can be administered in combination with thetreatments for the TBI or alone without any TBI treatment, depending onthe circumstances.

In embodiments of the above method, the method further comprisesmonitoring the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury.

In another embodiment, the present disclosure relates to a method oftreating a mild, moderate, severe, or moderate to severe traumatic braininjury in a human subject, the method comprising: a) performing an assayon a sample obtained from the subject within about 2 hours after anactual or suspected injury to the head to measure or detect a level ofcTnI and a level of an early biomarker, wherein the sample is abiological sample and the early biomarker comprises UCH-L1, GFAP, or acombination thereof; b) determining whether the subject has sustained amild or a moderate, severe, or moderate to severe traumatic brain injury(TBI), wherein the subject is determined as having (1) a moderate,severe, or a moderate to severe traumatic brain injury when the level ofcTnI in the sample is higher than a reference level of cTnI and thelevel of the early biomarker in the sample is higher than a referencelevel of the early biomarker or (2) a mild traumatic brain injury whenthe level of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker; and c) treating the subjectassessed as having a mild, moderate, severe, or moderate to severetraumatic brain injury with a traumatic brain injury treatment.

In some embodiments of the above method, the traumatic brain injurytreatment for a subject suffering from a mild TBI can involve having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. In other embodiments of the above method,the traumatic brain injury treatment for a subject suffering from amoderate, severe, or moderate to severe TBI, the treatment involves theadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt or providing of oneor more therapies (such as rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,etc.) or any combinations thereof. Optionally, such methods may alsoinvolve providing one or more cardioprotective therapies. Suchcardioprotective therapies can be administered in combination with thetreatments for the TBI or alone without any TBI treatment, depending onthe circumstances.

In embodiments of the above method, the method further comprisesmonitoring the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury.

In embodiments of any of the above described methods, the levels of cTnIis determined by an immunoassay.

In embodiments of any of the above described methods, the levels ofUCH-L1 is determined by an immunoassay

In embodiments of any of the above described methods, the levels of GFAPis determined by an immunoassay.

In embodiments of any of the above described methods, each of the levelsof cTnI and UCH-L1 are determined by immunoassay.

In embodiments of any of the above described methods, each of the levelsof cTnI and GFAP are determined by immunoassay.

In embodiments of any of the above described methods, each of the levelsof cTnI, GFAP and UCH-L1 are determined by immunoassay.

In embodiments of any of the above described methods, the levels of cTnIare determined by a clinical chemistry assay.

In embodiments of any of the above described methods, the levels ofUCH-L1 is determined by a clinical chemistry assay.

In embodiments of any of the above described methods, the levels of GFAPis determined by a clinical chemistry assay.

In embodiments of any of the above described methods, each of the levelsof cTnI and UCH-L1 are determined by a clinical chemistry assay.

In embodiments of any of the above described methods, each of the levelsof cTnI and GFAP are determined by a clinical chemistry assay.

In embodiments of any of the above described methods, each of the levelsof cTnI, GFAP and UCH-L1 are determined by a clinical chemistry assay.

In embodiments of any of the above described methods, the levels of cTnIare determined by a single molecule detection assay.

In embodiments of any of the above described methods, the levels ofUCH-L1 are determined by a single molecule detection assay.

In embodiments of any of the above described methods, the levels of GFAPare determined by a single molecule detection assay.

In embodiments of any of the above described methods, each of the levelsof cTnI and UCH-L1 are determined by a single molecule detection assay.

In embodiments of any of the above described methods, each of the levelsof cTnI and GFAP are determined by a single molecule detection assay.

In embodiments of any of the above described methods, each of the levelsof cTnI, GFAP and UCH-L1 are determined by single molecule detectionassay.

In embodiments of any of the above described methods, the sample is awhole blood sample.

In embodiments of any of the above described methods, the sample is aplasma sample.

In embodiments of any of the above described methods, the sample is aserum sample.

In embodiments of any of the above described methods, the measuring ofthe cTnI comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex,        -   such that a cTnI-capture antibody-cTnI            antigen-cTnI-detection antibody complex is formed, and    -   B. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

In embodiments of any of the above described methods, the sample isobtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

In embodiments of any of the above described methods, the sample isobtained after the subject has ingested or been exposed to a chemical,toxin or combination of a chemical and toxin. For example, the chemicalor toxin can be fire, mold, asbestos, a pesticide, an insecticide, anorganic solvent, a paint, a glue, a gas, an organic metal, a drug ofabuse or one or more combinations thereof.

In embodiments of any of the above described methods, the sample isobtained from a subject that suffers from an autoimmune disease, ametabolic disorder, a brain tumor, hypoxia, a virus, meningitis,hydrocephalus or combinations thereof.

In embodiments of any of the above described methods, the methods can becarried out on any subject without regard to factors selected from thegroup consisting of the subject's clinical condition, the subject'slaboratory values, the subject's classification as suffering from mild,moderate, severe, or a moderate to severe traumatic brain injury, thesubject's exhibition of low or high levels of cTnI, and the timing ofany event wherein said subject may have sustained an injury to the head.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows biomarker UCH-L1 result vs. time from injury.

FIG. 2 shows a box plot of UCH-L1 assay results by time point.

FIG. 3 shows a box plot of hsTnI assay results by time point.

FIG. 4 shows box plot of hsTnI assay results at Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1) correlated with positive vs.negative CT scan results.

FIG. 5 shows box plots of absolute amount (“absolute delta”) of hsTnIresults (i.e., the absolute difference between Time Point 2 (taken 3 to6 hours after the sample of Time Point 1) and Time Point 1 (taken within0 to 6 hours after head injury)) correlated with positive vs. negativeCT scan results.

FIG. 6 shows box plot of hsTnI assay results at Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 7 shows box plot of absolute amount (“absolute delta”) hsTnI assayresults (i.e., the absolute difference between Time Point 1 (takenwithin 0 to 6 hours after head injury) and Time Point 2 (taken 3 to 6hours after the sample of Time Point 1)) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 8 shows a receiver operating characteristic (ROC) analysis ofUCH-L1 assay results at Time Point 1 (taken within 0 to 12 hours afterhead injury) correlated with mild vs. moderate/severe TBI GCS scores.

FIG. 9 shows ROC analysis of UCH-L1 assay results at Time Point 1 (takenmore than 12 hours after head injury) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 10 shows ROC analysis of absolute amount (“absolute delta”) of thecombination of hsTnI levels and UCH-L1 levels (i.e., the absolutedifference between hsTnI levels at Time Point 2 and hsTnI levels at TimePoint 1 and the absolute difference between UCH-L1 levels at Time Point2 and UCH-L1 levels at Time Point 1) correlated with GCS Score result(mild vs. moderate/severe). The sample at Time Point 1 is taken within 0to 12 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 11 shows ROC analysis of absolute amount (“absolute delta”) of thecombination of hsTnI levels and UCH-L1 levels (i.e., the absolutedifference between hsTnI levels at Time Point 2 and hsTnI levels at TimePoint 1 and the absolute difference between UCH-L1 levels at Time Point2 and UCH-L1 levels at Time Point 1) correlated with GCS Score result(mild vs. moderate/severe). The sample at Time Point 1 is taken morethan 12 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 12 shows CT status (positive or negative CT scan results) and hsTnIlevels of human subjects vs. blood draw time relative to injury.

FIG. 13 shows GCS score results and hsTnI levels of human subjects vs.blood draw time relative to injury.

FIG. 14 shows CT status (positive or negative CT scan results) andubiquitin carboxy-terminal hydrolase L1 (UCH-L1) levels of humansubjects vs. blood draw time relative to injury.

FIG. 15 shows GCS score results and UCH-L1 levels of human subjects vs.blood draw time relative to injury.

FIG. 16 shows CT status (positive or negative CT scan results) and glialfibrillary acidic protein (GFAP) levels of human subjects vs. blood drawtime relative to injury.

FIG. 17 shows GCS score results and GFAP levels of human subjects vs.blood draw time relative to injury.

FIG. 18 shows a receiver operating characteristic (ROC) analysis ofhsTnI levels correlated with CT status (positive vs. negative CT scanresult) in samples taken within about 2 hours of suspected injury. Thesample at Time Point 1 is taken within 2 hours of head injury while thesample at Time Point 2 is taken about 3 to about 6 hours after the TimePoint 1 sample is taken.

FIG. 19 shows a receiver operating characteristic (ROC) analysis ofhsTnI levels correlated with mild vs. moderate/severe TBI GCS scores insamples taken within about 2 hours of suspected injury. The sample atTime Point 1 is taken within 2 hours of head injury while the sample atTime Point 2 is taken about 3 to about 6 hours after the Time Point 1sample is taken.

FIG. 20 shows ROC curve of hsTnI assay results for all of the subjectsat Time Point 1 correlated with positive vs. negative CT scan results.

FIG. 21 shows ROC curve of hsTnI assay results for all of the subjectsat Time Point 1 correlated with mild vs. moderate/severe TBI GCS scores.

FIG. 22 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with CT status(positive vs. negative CT scan result). The sample at Time Point 1 istaken within 2 hours of head injury while the sample at Time Point 2 istaken about 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 23 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with mild vs.moderate/severe TBI GCS scores. The sample at Time Point 1 is takenwithin 2 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken.

FIG. 24 shows ROC curve of absolute amount (“absolute delta”) hsTnIassay results for all of the subjects correlated with positive vs.negative CT scan results.

FIG. 25 shows ROC curve of absolute amount (“absolute delta”) hsTnIassay results for all of the subjects correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 26 shows ROC curve of UCH-L1 assay results for all of the subjectsat Time Point 1 correlated with positive vs. negative CT scan results.

FIG. 27 shows ROC curve of UCH-L1 assay results for all of the subjectsat Time Point 1 correlated with mild vs. moderate/severe TBI GCS scores.

FIG. 28 shows ROC analysis of the combination of hsTnI levels and UCH-L1levels correlated with CT status (positive vs. negative CT scan result)in samples taken at a first time point 1 within 24 hours after headinjury.

FIG. 29 shows ROC analysis of the combination of hsTnI levels and UCH-L1levels correlated with GCS Score result (mild vs. moderate/severe) insamples taken at a first time point 1 within 24 hours after head injury.

FIG. 30 shows ROC curve of GFAP assay results for all of the subjects atTime Point 1 correlated with positive vs. negative CT scan results.

FIG. 31 shows ROC curve of GFAP assay results for all of the subjects atTime Point 1 correlated with mild vs. moderate/severe TBI GCS scores.

FIG. 32 shows ROC analysis of the combination of hsTnI levels and GFAPlevels correlated with CT status (positive vs. negative CT scan result)in samples taken at a first time point 1 within 24 hours after headinjury.

FIG. 33 shows ROC analysis of the combination of hsTnI levels and GFAPlevels correlated with GCS Score result (mild vs. moderate/severe) insamples taken at a first time point 1 within 24 hours after head injury.

FIG. 34 shows a box plot of GFAP assay results by time point.

DETAILED DESCRIPTION

The present disclosure provides new and improved methods of using levelsand/or changes in the levels of cTnI (e.g., by performing an assay todetermine the level of cTnI in one or more biological samples and thencomparing those level(s) to one or more reference level(s)) as an aid inthe evaluation, diagnoses and/or stratification of whether a subjectthat has suffered an injury or is believed to have suffered an injury tothe head has suffered mild TBI, a moderate TBI, a severe TBI, a moderateto severe TBI or no TBI whatsoever. The methods described herein can beperformed quickly—in as little as 2 hours and up to about 24 hours afteran injury or suspected injury to the head. The use of cTnI todifferentiate between mild, moderate, severe, moderate to severe or noTBI in this manner is previously unknown. Not only do such methods allowa physician to quickly determine and classify (or reclassify) or triagea patient as having a TBI or no TBI, for those patients identified ordetermined to have suffered a TBI, the methods described herein allowthe physician to determine the type of TBI (mild versus moderate,severe, or moderate to severe). The ability to quickly determine whetherclassify a TBI as mild, moderate, severe or moderate to severe allowsthe physician to development an appropriate course of treatment (e.g.,treatment plan) for the subject. Such a treatment plan can includewhether to (1) order one or more additional tests to obtain furtherclinical information about the TBI (e.g., such as a MRI, etc.); (2)begin (continue) monitoring the subject; (3) begin treating the subjectwith a traumatic brain injury treatment (and if treatment is begun, whattype of treatment to begin (e.g, one or more therapeutic treatments,protecting the airway, one or more surgical treatments, ordering rest,etc.); (4) begin any cardioprotective treatment to protect the heart ofthe subject (such as, optionally, by the administration of one or morebeta-blockers, diuretics, angiotensin-converting inhibitors, calciumchannel blockers, lipid lowering therapies, statins, nitrates,antiplatelet therapy, anticlotting agents, anticoagulation agents orcombinations thereof, or other cardioprotective agents known in theart); or (5) perform any combinations of (1)-(4).

Additionally, the present disclosure provides methods of using levels orchanges in the levels of cTnI and UCH-L1 and/or GFAP as an aid indetermining whether a head computerized tomography (CT) should beperformed on a subject that has suffered or is believed to have suffereda TBI. The methods described herein can be performed quickly—in aslittle as 2 hours and up to about 24 hours after an injury or suspectedinjury to the head. The use of cTnI and UCH-L1 and/or GFAP as an aid inassisting a physician to determine whether or to perform a head CT insubjects that have suffered or believed to have suffered a TBI ispreviously not known.

Additionally, the present disclosure provides methods of treating atraumatic brain injury. Specifically, the methods involve using levelsand/or changes in the levels of cTnI and UCH-L1 and/or GFAP as describedherein (e.g., by performing an assay to determine the level of cTnI andUCH-L1 and/or GFAP in one or more biological samples and then comparingthose level(s) to one or more reference level(s)) to evaluate, diagnoseand/or stratify whether a subject that has suffered an injury to thehead or is believed to have suffered an injury to the head has sufferedmild TBI, a moderate TBI, severe TBI, moderate to severe TBI or no TBI.Once a subject has been identified, determined, classified or stratifiedas having a mild TBI or a moderate, severe or moderate to severe TBI,then depending on the type of TBI (mild versus moderate, severe ormoderate to severe), the subject can be treated with an appropriatetraumatic brain injury treatment. For example, for a mild TBI, thetraumatic brain injury treatment may involve one or more of having thesubject rest for a certain period of time, abstain from physicalactivities for a certain period of time, administration of one or moretherapeutics (e.g., drugs to provide relief for a headache or migraine,etc.) or combinations thereof. For a moderate, severe, or moderate tosevere TBI, the traumatic brain injury treatment may involveadministration of one or more therapeutics (e.g., drugs such asdiuretics, anti-seizure drugs), performing one or more surgicalprocedures (e.g., such as removal of a hematoma, repairing a skullfracture, decompressive crainiectomy, etc.), receipt of one or moretherapies (such as rehabilitation, physical therapy, occupationaltherapy, cognitive behavioral therapy, anger management, etc.) orcombinations thereof. Optionally, such methods may also involveproviding one or more cardioprotective therapies. Such cardioprotectivetherapies can be administered in combination with the treatments for theTBI or alone without any TBI treatment, depending on the circumstances.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI).Furthermore, in order to confirm that the changes in amounts or levelscTnI in the methods described herein are attributable to a head injuryor a suspected injury to the head of a subject and not the result of anacute cardiac syndrome (such as a myocardial infarction, heart failure,etc.), a physician or other healthcare provider could conduct or performone or more additional tests or procedures to confirm the absence of anacute cardiac syndrome. Such additional tests or procedures include oneor more of an electrocardiogram, a complete blood cell (CBC) count, acomprehensive metabolic panel, a lipid profile (e.g., to determine HDL,LDL, triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

Section headings as used in this section and the entire disclosureherein are merely for organizational purposes and are not intended to belimiting.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentdisclosure. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity matured antibody” is used herein to refer to an antibody withone or more alterations in one or more CDRs, which result in animprovement in the affinity (i.e., K_(D), k_(d) or k_(a)) of theantibody for a target antigen compared to a parent antibody, which doesnot possess the alteration(s). Exemplary affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.A variety of procedures for producing affinity matured antibodies isknown in the art, including the screening of a combinatory antibodylibrary that has been prepared using bio-display. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); and Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity-enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

“Antibody” and “antibodies” as used herein refers to monoclonalantibodies, multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized), animal antibodies such as,but not limited to, a bird (for example, a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate(for example, a monkey, a chimpanzee, etc.), recombinant antibodies,chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments, F(ab′)₂fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11):1290-1297 (2007) and PCT International ApplicationWO 2001/058956, the contents of each of which are herein incorporated byreference), and functionally active epitope-binding fragments of any ofthe above. In particular, antibodies include immunoglobulin moleculesand immunologically active fragments of immunoglobulin molecules,namely, molecules that contain an analyte-binding site. Immunoglobulinmolecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, andIgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), orsubclass. For simplicity sake, an antibody against an analyte isfrequently referred to herein as being either an “anti-analyte antibody”or merely an “analyte antibody” (e.g., an anti-cardiac troponin I and/oranti-UCH-L1 and/or anti-GFAP antibody or a cardiac troponin I and/oranti-UCH-L1 and/or anti-GFAP antibody).

“Antibody fragment” as used herein refers to a portion of an intactantibody comprising the antigen-binding site or variable region. Theportion does not include the constant heavy chain domains (i.e., CH2,CH3, or CH4, depending on the antibody isotype) of the Fc region of theintact antibody. Examples of antibody fragments include, but are notlimited to, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)₂fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv)molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs ofthe light-chain variable domain, single-chain polypeptides containingonly one heavy chain variable region, and single-chain polypeptidescontaining the three CDRs of the heavy chain variable region.

The “area under curve” or “AUC” refers to area under a ROC curve. AUCunder a ROC curve is a measure of accuracy. An AUC of 1 represents aperfect test, whereas an AUC of 0.5 represents an insignificant test. Apreferred AUC may be at least approximately 0.700, at leastapproximately 0.750, at least approximately 0.800, at leastapproximately 0.850, at least approximately 0.900, at leastapproximately 0.910, at least approximately 0.920, at leastapproximately 0.930, at least approximately 0.940, at leastapproximately 0.950, at least approximately 0.960, at leastapproximately 0.970, at least approximately 0.980, at leastapproximately 0.990, or at least approximately 0.995.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support. One example of a bead or particleis a microparticle. Microparticles that can be used herein can be anytype known in the art. For example, the bead or particle can be amagnetic bead or magnetic particle. Magnetic beads/particles may beferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The microparticles can be of any size thatwould work in the methods described herein, e.g., from about 0.75 toabout 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3nm.

“Binding protein” is used herein to refer to a monomeric or multimericprotein that binds to and forms a complex with a binding partner, suchas, for example, a polypeptide, an antigen, a chemical compound or othermolecule, or a substrate of any kind. A binding protein specificallybinds a binding partner. Binding proteins include antibodies, as well asantigen-binding fragments thereof and other various forms andderivatives thereof as are known in the art and described herein below,and other molecules comprising one or more antigen-binding domains thatbind to an antigen molecule or a particular site (epitope) on theantigen molecule. Accordingly, a binding protein includes, but is notlimited to, an antibody a tetrameric immunoglobulin, an IgG molecule, anIgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

“Bispecific antibody” is used herein to refer to a full-length antibodythat is generated by quadroma technology (see Milstein et al., Nature,305(5934): 537-540 (1983)), by chemical conjugation of two differentmonoclonal antibodies (see, Staerz et al., Nature, 314(6012): 628-631(1985)), or by knob-into-hole or similar approaches, which introducemutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci.USA, 90(14): 6444-6448 (1993)), resulting in multiple differentimmunoglobulin species of which only one is the functional bispecificantibody. A bispecific antibody binds one antigen (or epitope) on one ofits two binding arms (one pair of HC/LC), and binds a different antigen(or epitope) on its second arm (a different pair of HC/LC). By thisdefinition, a bispecific antibody has two distinct antigen-binding arms(in both specificity and CDR sequences), and is monovalent for eachantigen to which it binds to.

As used herein, the terms “cardiac troponin I”, “cTnI” or “troponin I”as used interchangeably herein, refers to one of two unique forms ofcardiac troponin (the other unique form being cardiac troponin T (alsoreferred to as “cTnT”)), released into the blood from cardiac muscle forwhich several species may exist in the blood. Not only does the term“cardiac troponin I” or “cTnI” include the full-length version of thisform but it also includes: (1) various complexes of cTnI (namely, witheach other and/or with cardiac troponin C (cTnC)); (2) fragments of cTnIwhich result from proteolytic degradation; (3) phosphorylated andoxidized forms of cTnI (See, for example, U.S. Pat. No. 6,991,907, thecontents of which are herein incorporated by reference); and (4) anyisoforms of cTnI.

In some embodiments, the methods of the present disclosure allow for thedetection and/or determination of concentration of one or more of thevarious forms of cTnI in a sample as a separate entity, e.g., complexedcTnI, free cTnI (e.g., such as fully length, fragments, isoforms, etc.),muddied cTnI (e.g., oxidized or phosphorylated), and, optionally,provides a concentration for the cTnI in the biological sample.

More specifically, in some embodiments, the disclosure described hereinemploys highly sensitivity assays that allow for the detection andquantification of cTnI at levels 10- to 100-fold lower than levelsmeasured by traditional troponin assays (e.g., immunoassays) known inthe art. More specifically, assays are defined as high sensitivity(e.g., high sensitivity assays for troponin) if such assays meet atleast the following two conditions: 1) a coefficient of variance lessthan 10% at the 99th percentile value of the reference healthypopulation and 2) concentrations above the assay's limit of detectionare measurable in greater than 50% of healthy individuals (See, Apple FS, et al., Clin Chem., 58:54-61 (2012), the contents of which are herebyincorporated by reference). Examples of assays known in the art thatallow for the high-sensitive detection of troponin include thoseavailable from Quanterix (Simoa Human Troponin-I immunoassay) forresearch use only as well as those described in U.S. Pat. No. 9,182,405,the contents of which are hereby incorporated by reference.

“Cardiac Troponin I status” or “cTnI status” as used interchangeablyherein can mean either the level or amount of cardiac troponin I at apoint in time (such as with a single measure of troponin I), the levelor amount of cardiac troponin I associated with monitoring (such as witha repeat test on a subject to identify an increase or decrease incardiac troponin I amount), the level or amount of cardiac troponin Iassociated with treatment for traumatic brain injury (whether a primarybrain injury and/or a secondary brain injury) or combinations thereof.

“CDR” is used herein to refer to the “complementarity determiningregion” within an antibody variable sequence. There are three CDRs ineach of the variable regions of the heavy chain and the light chain.Proceeding from the N-terminus of a heavy or light chain, these regionsare denoted “CDR1”, “CDR2”, and “CDR3”, for each of the variableregions. The term “CDR set” as used herein refers to a group of threeCDRs that occur in a single variable region that binds the antigen. Anantigen-binding site, therefore, may include six CDRs, comprising theCDR set from each of a heavy and a light chain variable region. Apolypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) maybe referred to as a “molecular recognition unit.” Crystallographicanalyses of antigen-antibody complexes have demonstrated that the aminoacid residues of CDRs form extensive contact with bound antigen, whereinthe most extensive antigen contact is with the heavy chain CDR3. Thus,the molecular recognition units may be primarily responsible for thespecificity of an antigen-binding site. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as “Kabat CDRs”.Chothia and coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917(1987); and Chothia et al., Nature, 342: 877-883 (1989)) found thatcertain sub-portions within Kabat CDRs adopt nearly identical peptidebackbone conformations, despite having great diversity at the level ofamino acid sequence. These sub-portions were designated as “L1”, “L2”,and “L3”, or “H1”, “H2”, and “H3”, where the “L” and the “H” designatethe light chain and the heavy chain regions, respectively. These regionsmay be referred to as “Chothia CDRs”, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995),and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDRboundary definitions may not strictly follow one of the herein systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat- or Chothia-defined CDRs.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection or conjugate a calibrator, a control,a sensitivity panel, a container, a buffer, a diluent, a salt, anenzyme, a co-factor for an enzyme, a detection reagent, a pretreatmentreagent/solution, a substrate (e.g., as a solution), a stop solution,and the like that can be included in a kit for assay of a test sample,such as a patient urine, whole blood, serum or plasma sample, inaccordance with the methods described herein and other methods known inthe art. Some components can be in solution or lyophilized forreconstitution for use in an assay.

“Correlated to” as used herein refers to compared to.

“CT scan” as used herein refers to a computerized tomography (CT) scan.A CT scan combines a series of X-ray images taken from different anglesand uses computer processing to create cross-sectional images, orslices, of the bones, blood vessels and soft tissues inside your body.The CT scan may use X-ray CT, positron emission tomography (PET),single-photon emission computed tomography (SPECT), computed axialtomography (CAT scan), or computer aided tomography. The CT scan may bea conventional CT scan or a spiral/helical CT scan. In a conventional CTscan, the scan is taken slice by slice and after each slice the scanstops and moves down to the next slice, e.g., from the top of theabdomen down to the pelvis. The conventional CT scan requires patientsto hold their breath to avoid movement artefact. The spiral/helical CTscan is a continuous scan which is taken in a spiral fashion and is amuch quicker process where the scanned images are contiguous.

“Derivative” of an antibody as used herein may refer to an antibodyhaving one or more modifications to its amino acid sequence whencompared to a genuine or parent antibody and exhibit a modified domainstructure. The derivative may still be able to adopt the typical domainconfiguration found in native antibodies, as well as an amino acidsequence, which is able to bind to targets (antigens) with specificity.Typical examples of antibody derivatives are antibodies coupled to otherpolypeptides, rearranged antibody domains, or fragments of antibodies.The derivative may also comprise at least one further compound, e.g., aprotein domain, said protein domain being linked by covalent ornon-covalent bonds. The linkage can be based on genetic fusion accordingto the methods known in the art. The additional domain present in thefusion protein comprising the antibody may preferably be linked by aflexible linker, advantageously a peptide linker, wherein said peptidelinker comprises plural, hydrophilic, peptide-bonded amino acids of alength sufficient to span the distance between the C-terminal end of thefurther protein domain and the N-terminal end of the antibody or viceversa. The antibody may be linked to an effector molecule having aconformation suitable for biological activity or selective binding to asolid support, a biologically active substance (e.g., a cytokine orgrowth hormone), a chemical agent, a peptide, a protein, or a drug, forexample.

“Determined by an assay” is used herein to refer to the determination ofa reference level by any appropriate assay. The determination of areference level may, in some embodiments, be achieved by an assay of thesame type as the assay that is to be applied to the sample from thesubject (for example, by an immunoassay, clinical chemistry assay, asingle molecule detection assay, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS)). The determination of a reference level may, in someembodiments, be achieved by an assay of the same type and under the sameassay conditions as the assay that is to be applied to the sample fromthe subject. As noted herein, this disclosure provides exemplaryreference levels (e.g., calculated by comparing reference levels atdifferent time points). It is well within the ordinary skill of one inthe art to adapt the disclosure herein for other assays to obtainassay-specific reference levels for those other assays based on thedescription provided by this disclosure. For example, a set of trainingsamples comprising samples obtained from human subjects known to havesustained an injury to the head (and more particularly, samples obtainedfrom human subjects known to have sustained a (i) mild TBI; and/or (ii)moderate, severe, or moderate to severe TBI and samples obtained fromhuman subjects known not to have sustained an injury to the head may beused to obtain assay-specific reference levels. It will be understoodthat a reference level “determined by an assay” and having a recitedlevel of “sensitivity” and/or “specificity” is used herein to refer to areference level which has been determined to provide a method of therecited sensitivity and/or specificity when said reference level isadopted in the methods of the disclosure. It is well within the ordinaryskill of one in the art to determine the sensitivity and specificityassociated with a given reference level in the methods of thedisclosure, for example by repeated statistical analysis of assay datausing a plurality of different possible reference levels.

Practically, when discriminating between a subject as having a traumaticbrain injury or not having a traumatic brain injury or a subject ashaving a mild versus a moderate, severe, or moderate to severe traumaticbrain injury, the skilled person will balance the effect of raising acutoff on sensitivity and specificity. Raising or lowering a cutoff willhave a well-defined and predictable impact on sensitivity andspecificity, and other standard statistical measures. It is well knownthat raising a cutoff will improve specificity but is likely to worsensensitivity (proportion of those with disease who test positive). Incontrast, lowering a cutoff will improve sensitivity but will worsenspecificity (proportion of those without disease who test negative). Theramifications for detecting traumatic brain injury or determining a mildversus moderate, severe, or moderate to severe traumatic brain injurywill be readily apparent to those skilled in the art. In discriminatingwhether a subject has or does not have a traumatic brain injury or amild versus a moderate, severe, or moderate to severe traumatic braininjury, the higher the cutoff, specificity improves as more truenegatives (i.e., subjects not having a traumatic brain injury, nothaving a mild traumatic brain injury, not have a moderate traumaticbrain injury, not having a severe traumatic brain injury or not having amoderate, severe, or moderate to severe traumatic brain injury) aredistinguished from those having a traumatic brain injury, a mildtraumatic brain injury, a moderate traumatic brain injury, a severetraumatic brain injury or a moderate, severe, or moderate to severetraumatic brain injury. But at the same time, raising the cutoffdecreases the number of cases identified as positive overall, as well asthe number of true positives, so the sensitivity must decrease.Conversely, the lower the cutoff, sensitivity improves as more truepositives (i.e., subjects having a traumatic brain injury, having a mildtraumatic brain injury, having a moderate traumatic brain injury, havinga severe traumatic brain injury or having a moderate, severe, ormoderate to severe traumatic brain injury) are distinguished from thosewho do not have a traumatic brain injury, a mild traumatic brain injure,a moderate traumatic brain injury, a severe traumatic brain injury or amoderate, severe, or moderate to severe traumatic brain injury. But atthe same time, lowering the cutoff increases the number of casesidentified as positive overall, as well as the number of falsepositives, so the specificity must decrease.

Generally, a high sensitivity value helps one of skill rule out diseaseor condition (such as a traumatic brain injury, mild traumatic braininjury, moderate traumatic brain injury, severe traumatic brain injuryor moderate to severe traumatic brain injury), and a high specificityvalue helps one of skill rule in disease or condition. Whether one ofskill desires to rule out or rule in disease depends on what theconsequences are for the patient for each type of error. Accordingly,one cannot know or predict the precise balancing employed to derive atest cutoff without full disclosure of the underlying information on howthe value was selected. The balancing of sensitivity against specificityand other factors will differ on a case-by-case basis. This is why it issometimes preferable to provide alternate cutoff (e.g., reference)values so a physician or practitioner can choose.

“Drugs of abuse” is used herein to refer to one or more additivesubstances (such as a drug) taken for non-medical reasons (such as for,example, recreational and/or mind-altering effects). Excessiveoverindulgence, use or dependence of such drugs of abuse is oftenreferred to as “substance abuse”. Examples of drugs of abuse includealcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens(such as ketamine, mescaline (peyote), PCP, psilocybin, DMT and/or LSD),methaqualone, opioids, amphetamines (including methamphetamines),anabolic steroids, inhalants (namely, substances which contain volatilesubstances that contain psychoactive properties such as, for example,nitrites, spray paints, cleaning fluids, markers, glues, etc.) andcombinations thereof.

“Dual-specific antibody” is used herein to refer to a full-lengthantibody that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly, a dual-specific binding protein has twoidentical antigen binding arms, with identical specificity and identicalCDR sequences, and is bivalent for each antigen to which it binds.

“Dual variable domain” is used herein to refer to two or more antigenbinding sites on a binding protein, which may be divalent (two antigenbinding sites), tetravalent (four antigen binding sites), or multivalentbinding proteins. DVDs may be monospecific, i.e., capable of binding oneantigen (or one specific epitope), or multispecific, i.e., capable ofbinding two or more antigens (i.e., two or more epitopes of the sametarget antigen molecule or two or more epitopes of different targetantigens). A preferred DVD binding protein comprises two heavy chain DVDpolypeptides and two light chain DVD polypeptides and is referred to asa “DVD immunoglobulin” or “DVD-Ig.” Such a DVD-Ig binding protein isthus tetrameric and reminiscent of an IgG molecule, but provides moreantigen binding sites than an IgG molecule. Thus, each half of atetrameric DVD-Ig molecule is reminiscent of one half of an IgG moleculeand comprises a heavy chain DVD polypeptide and a light chain DVDpolypeptide, but unlike a pair of heavy and light chains of an IgGmolecule that provides a single antigen binding domain, a pair of heavyand light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of a DVD-Ig binding protein may be derivedfrom a donor (“parental”) monoclonal antibody and thus comprises a heavychain variable domain (VH) and a light chain variable domain (VL) with atotal of six CDRs involved in antigen binding per antigen binding site.Accordingly, a DVD-Ig binding protein that binds two different epitopes(i.e., two different epitopes of two different antigen molecules or twodifferent epitopes of the same antigen molecule) comprises an antigenbinding site derived from a first parental monoclonal antibody and anantigen binding site of a second parental monoclonal antibody.

A description of the design, expression, and characterization of DVD-Igbinding molecules is provided in PCT Publication No. WO 2007/024715,U.S. Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297(2007). A preferred example of such DVD-Ig molecules comprises a heavychain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a heavy chain constant domain, X1 is alinker with the proviso that it is not CH1, X2 is an Fc region, and n is0 or 1, but preferably 1; and a light chain that comprises thestructural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain, X1 is a linker with the proviso that itis not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, butpreferably 1. Such a DVD-Ig may comprise two such heavy chains and twosuch light chains, wherein each chain comprises variable domains linkedin tandem without an intervening constant region between variableregions, wherein a heavy chain and a light chain associate to formtandem functional antigen binding sites, and a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1, VD2, VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate to formthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein not only binds thesame target molecules bound by its parental monoclonal antibodies, butalso possesses one or more desirable properties of one or more of itsparental monoclonal antibodies. Preferably, such an additional propertyis an antibody parameter of one or more of the parental monoclonalantibodies. Antibody parameters that may be contributed to a DVD-Igbinding protein from one or more of its parental monoclonal antibodiesinclude, but are not limited to, antigen specificity, antigen affinity,potency, biological function, epitope recognition, protein stability,protein solubility, production efficiency, immunogenicity,pharmacokinetics, bioavailability, tissue cross reactivity, andorthologous antigen binding.

A DVD-Ig binding protein binds at least one epitope of cardiac troponinI and/or UCH-L1 and/or GFAP. Non-limiting examples of a DVD-Ig bindingprotein include a DVD-Ig binding protein that binds one or more epitopesof cardiac troponin I and/or UCH-L1 and/or GFAP, a DVD-Ig bindingprotein that binds an epitope of a human cardiac troponin I and/orUCH-L1 and/or GFAP and an epitope of cardiac troponin I and/or UCH-L1and/or GFAP of another species (for example, mouse), and a DVD-Igbinding protein that binds an epitope of a human cardiac troponin Iand/or UCH-L1 and/or GFAP and an epitope of another target molecule.

“Dynamic range” as used herein refers to range over which an assayreadout is proportional to the amount of target molecule or analyte inthe sample being analyzed.

“Epitope,” or “epitopes,” or “epitopes of interest” refer to a site(s)on any molecule that is recognized and can bind to a complementarysite(s) on its specific binding partner. The molecule and specificbinding partner are part of a specific binding pair. For example, anepitope can be on a polypeptide, a protein, a hapten, a carbohydrateantigen (such as, but not limited to, glycolipids, glycoproteins orlipopolysaccharides), or a polysaccharide. Its specific binding partnercan be, but is not limited to, an antibody.

“Fragment antigen-binding fragment” or “Fab fragment” as used hereinrefers to a fragment of an antibody that binds to antigens and thatcontains one antigen-binding site, one complete light chain, and part ofone heavy chain. Fab is a monovalent fragment consisting of the VL, VH,CL and CH1 domains. Fab is composed of one constant and one variabledomain of each of the heavy and the light chain. The variable domaincontains the paratope (the antigen-binding site), comprising a set ofcomplementarity determining regions, at the amino terminal end of themonomer. Each arm of the Y thus binds an epitope on the antigen. Fabfragments can be generated such as has been described in the art, e.g.,using the enzyme papain, which can be used to cleave an immunoglobulinmonomer into two Fab fragments and an Fc fragment, or can be produced byrecombinant means.

“F(ab′)₂ fragment” as used herein refers to antibodies generated bypepsin digestion of whole IgG antibodies to remove most of the Fc regionwhile leaving intact some of the hinge region. F(ab′)₂ fragments havetwo antigen-binding F(ab) portions linked together by disulfide bonds,and therefore are divalent with a molecular weight of about 110 kDa.Divalent antibody fragments (F(ab′)₂ fragments) are smaller than wholeIgG molecules and enable a better penetration into tissue thusfacilitating better antigen recognition in immunohistochemistry. The useof F(ab′)₂ fragments also avoids unspecific binding to Fc receptor onlive cells or to Protein A/G. F(ab′)₂ fragments can both bind andprecipitate antigens.

“Framework” (FR) or “Framework sequence” as used herein may mean theremaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems (for example, see above), the meaning of a framework sequence issubject to correspondingly different interpretations. The six CDRs(CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavychain) also divide the framework regions on the light chain and theheavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on eachchain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2and FR3, and CDR3 between FR3 and FR4. Without specifying the particularsub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred byothers, represents the combined FRs within the variable region of asingle, naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain FR sequences are known in the art thatcan be used as heavy chain and light chain “acceptor” frameworksequences (or simply, “acceptor” sequences) to humanize a non-humanantibody using techniques known in the art. In one embodiment, humanheavy chain and light chain acceptor sequences are selected from theframework sequences listed in publicly available databases such asV-base (hypertext transfer protocol://vbase.mrc-cpe.cam.ac.uk/) or inthe international ImMunoGeneTics® (IMGT®) immunogenetics informationsystem (hypertext transferprotocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).

“Functional antigen binding site” as used herein may mean a site on abinding protein (e.g., an antibody) that is capable of binding a targetantigen. The antigen binding affinity of the antigen binding site maynot be as strong as the parent binding protein, e.g., parent antibody,from which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating protein, e.g., antibody, binding to an antigen. Moreover,the antigen binding affinity of each of the antigen binding sites of amultivalent protein, e.g., multivalent antibody, herein need not bequantitatively the same.

“GFAP” is used herein to describe glial fibrillary acidic protein. GFAPis a protein that is encoded by the GFAP gene in humans, and which canbe produced (e.g., by recombinant means, in other species).

“GFAP status” can mean either the level or amount of GFAP at a point intime (such as with a single measure of GFAP), the level or amount ofGFAP associated with monitoring (such as with a repeat test on a subjectto identify an increase or decrease in GFAP amount), the level or amountof GFAP associated with treatment for traumatic brain injury (whether aprimary brain injury and/or a secondary brain injury) or combinationsthereof.

“Glasgow Coma Scale” or “GCS” as used herein refers to a 15 point scalefor estimating and categorizing the outcomes of brain injury on thebasis of overall social capability or dependence on others. The testmeasures the motor response, verbal response and eye opening responsewith these values: I. Motor Response (6—Obeys commands fully;5—Localizes to noxious stimuli; 4—Withdraws from noxious stimuli;3—Abnormal flexion, i.e., decorticate posturing; 2—Extensor response,i.e., decerebrate posturing; and 1—No response); II. Verbal Response(5—Alert and Oriented; 4—Confused, yet coherent, speech; 3—Inappropriatewords and jumbled phrases consisting of words; 2—Incomprehensiblesounds; and 1—No sounds); and III. Eye Opening (4—Spontaneous eyeopening; 3—Eyes open to speech; 2—Eyes open to pain; and 1—No eyeopening). The final score is determined by adding the values ofI+II+III. The final score can be categorized into four possible levelsfor survival, with a lower number indicating a more severe injury and apoorer prognosis: Mild (13-15); Moderate Disability (9-12) (Loss ofconsciousness greater than 30 minutes; Physical or cognitive impairmentswhich may or may resolve: and Benefit from Rehabilitation); SevereDisability (3-8) (Coma: unconscious state. No meaningful response, novoluntary activities); and Vegetative State (Less Than 3) (Sleep wakecycles; Arousal, but no interaction with environment; No localizedresponse to pain). Moderate brain injury is defined as a brain injuryresulting in a loss of consciousness from 20 minutes to 6 hours and aGlasgow Coma Scale of 9 to 12. Severe brain injury is defined as a braininjury resulting in a loss of consciousness of greater than 6 hours anda Glasgow Coma Scale of 3 to 8.

“Glasgow Outcome Scale” as used herein refers to a global scale forfunctional outcome that rates patient status into one of fivecategories: Dead, Vegetative State, Severe Disability, ModerateDisability or Good Recovery.

“Extended Glasgow Outcome Scale” or “GOSE” as used interchangeablyherein provides more detailed categorization into eight categories bysubdividing the categories of severe disability, moderate disability andgood recovery into a lower and upper category as shown in Table 1.

TABLE 1 1 Death D 2 Vegetative VX Condition of unawareness with onlyreflex state responses but with periods of spontaneous eye opening 3Lower SD − Patient who is dependent for daily support for severe mentalor physical disability, usually a disability combination of both. If thepatient can be left 4 Upper severe SD + alone for more than 8 hours athome it is upper disability level of SD, if not then it is low level ofSD. 5 Lower MD − Patients have some disability such as aphasia, moderatehemiparesis or epilepsy and/or deficits of disability memory orpersonality but are able to look after 6 Upper MD + themselves. They areindependent at home but moderate dependent outside. If they are able toreturn to disability work even with special arrangement it is upperlevel of MD, if not then it is low level of MD. 7 Lower GR − Resumptionof normal life with the capacity to good GR + work even if pre-injurystatus has not been recovery achieved. Some patients have minor 8 Upperneurological or psychological deficits. If these good deficits are notdisabling then it is upper level recovery of GR, if disabling then it islower level of GR.

“Humanized antibody” is used herein to describe an antibody thatcomprises heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or VL sequence has been altered to be more “human-like,” i.e.,more similar to human germline variable sequences. A “humanizedantibody” is an antibody or a variant, derivative, analog, or fragmentthereof, which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of a non-human antibody CDR. A humanizedantibody comprises substantially all of at least one, and typically two,variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin (i.e., donor antibody) and all or substantially all ofthe framework regions are those of a human immunoglobulin consensussequence. In an embodiment, a humanized antibody also comprises at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. In some embodiments, a humanized antibody containsthe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

A humanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE, and any isotype, includingwithout limitation IgG1, IgG2, IgG3, and IgG4. A humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well-known in the art.

The framework regions and CDRs of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion, and/or deletion of at least one amino acid residue so thatthe CDR or framework residue at that site does not correspond to eitherthe donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence.

“Hyperacute” as used herein refers to extremely acute or within a courseof about 2 hours of the injury or suspected injury to the head.Hyperacute is within an early stage, e.g., a hyperacute biomarker is anearly biomarker, such as cTnI, ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof, that can be used to assess injury or suspected injury withinthe early stage of about 2 hours of injury or suspected injury.

“Identical” or “identity,” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are the same over aspecified region. The percentage can be calculated by optimally aligningthe two sequences, comparing the two sequences over the specifiedregion, determining the number of positions at which the identicalresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the specified region, and multiplying the result by 100to yield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of the single sequence are included in thedenominator but not the numerator of the calculation.

“Injury to the head” or “head injury” as used interchangeably herein,refers to any trauma to the scalp, skull, or brain. Such injuries mayinclude only a minor bump on the skull or may be a serious brain injury.Such injuries include primary injuries to the brain and/or secondaryinjuries to the brain. Primary brain injuries occur during the initialinsult and result from displacement of the physical structures of thebrain. More specifically, a primary brain injury is the physical damageto parenchyma (tissue, vessels) that occurs during the traumatic event,resulting in shearing and compression of the surrounding brain tissue.Secondary brain injuries occur subsequent to the primary injury and mayinvolve an array of cellular processes. More specifically, a secondarybrain injury refers to the changes that evolve over a period of time(from hours to days) after the primary brain injury. It includes anentire cascade of cellular, chemical, tissue, or blood vessel changes inthe brain that contribute to further destruction of brain tissue.

An injury to the head can be either closed or open (penetrating). Aclosed head injury refers to a trauma to the scalp, skull or brain wherethere is no penetration of the skull by a striking object. An open headinjury refers a trauma to the scalp, skull or brain where there ispenetration of the skull by a striking object. An injury to the head maybe caused by physical shaking of a person, by blunt impact by anexternal mechanical or other force that results in a closed or open headtrauma (e.g., vehicle accident such as with an automobile, plane, train,etc.; blow to the head such as with a baseball bat, or from a firearm),a cerebral vascular accident (e.g., stroke), one or more falls (e.g., asin sports or other activities), explosions or blasts (collectively,“blast injuries”) and by other types of blunt force trauma.Alternatively, an injury to the head may be caused by the ingestionand/or exposure to a chemical, toxin or a combination of a chemical andtoxin. Examples of such chemicals and/or toxins include fires, molds,asbestos, pesticides and insecticides, organic solvents, paints, glues,gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organicmetals (such as methyl mercury, tetraethyl lead and organic tin) and/orone or more drugs of abuse. Alternatively, an injury to the head may becaused as a result of a subject suffering from an autoimmune disease, ametabolic disorder, a brain tumor, one or more viruses, meningitis,hydrocephalus, hypoxia or any combinations thereof. In some cases, it isnot possible to be certain whether any such event or injury has occurredor taken place. For example, there may be no history on a patient orsubject, the subject may be unable to speak, the subject may be aware ofwhat events they were exposed to, etc. Such circumstances are describedherein as the subject “may have sustained an injury to the head.” Incertain embodiments herein, the closed head injury does not include andspecifically excludes a cerebral vascular accident, such as stroke.

“Isolated polynucleotide” as used herein may mean a polynucleotide(e.g., of genomic, cDNA, or synthetic origin, or a combination thereof)that, by virtue of its origin, the isolated polynucleotide is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

“Label” and “detectable label” as used herein refer to a moiety attachedto an antibody or an analyte to render the reaction between the antibodyand the analyte detectable, and the antibody or analyte so labeled isreferred to as “detectably labeled.” A label can produce a signal thatis detectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromagens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like.Representative examples of labels include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety, itself, may not be detectable but may become detectable uponreaction with yet another moiety. Use of the term “detectably labeled”is intended to encompass such labeling.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as 3H,14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzymatic label (such as horseradish peroxidase, alkalineperoxidase, glucose 6-phosphate dehydrogenase, and the like), achemiluminescent label (such as acridinium esters, thioesters, orsulfonamides; luminol, isoluminol, phenanthridinium esters, and thelike), a fluorescent label (such as fluorescein (e.g., 5-fluorescein,6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein,6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluoresceinisothiocyanate, and the like)), rhodamine, phycobiliproteins,R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmiumselenide), a thermometric label, or an immuno-polymerase chain reactionlabel. An introduction to labels, labeling procedures and detection oflabels is found in Polak and Van Noorden, Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and inHaugland, Handbook of Fluorescent Probes and Research Chemicals (1996),which is a combined handbook and catalogue published by MolecularProbes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA(see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,and 5,352,803, which are hereby incorporated by reference in theirentireties). An acridinium compound can be used as a detectable label ina homogeneous chemiluminescent assay (see, e.g., Adamczyk et al.,Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg.Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem.Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782(2003)).

In one aspect, the acridinium compound is an acridinium-9-carboxamide.Methods for preparing acridinium 9-carboxamides are described inMattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al.,J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999);Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly etal., In Luminescence Biotechnology: Instruments and Applications; Dyke,K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524and 5,783,699 (each of which is incorporated herein by reference in itsentirety for its teachings regarding same).

Another example of an acridinium compound is an acridinium-9-carboxylatearyl ester. An example of an acridinium-9-carboxylate aryl ester offormula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate(available from Cayman Chemical, Ann Arbor, Mich.). Methods forpreparing acridinium 9-carboxylate aryl esters are described in McCapraet al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244(2000); and U.S. Pat. No. 5,241,070 (each of which is incorporatedherein by reference in its entirety for its teachings regarding same).Such acridinium-9-carboxylate aryl esters are efficient chemiluminescentindicators for hydrogen peroxide produced in the oxidation of an analyteby at least one oxidase in terms of the intensity of the signal and/orthe rapidity of the signal. The course of the chemiluminescent emissionfor the acridinium-9-carboxylate aryl ester is completed rapidly, i.e.,in under 1 second, while the acridinium-9-carboxamide chemiluminescentemission extends over 2 seconds. Acridinium-9-carboxylate aryl ester,however, loses its chemiluminescent properties in the presence ofprotein. Therefore, its use requires the absence of protein duringsignal generation and detection. Methods for separating or removingproteins in the sample are well-known to those skilled in the art andinclude, but are not limited to, ultrafiltration, extraction,precipitation, dialysis, chromatography, and/or digestion (see, e.g.,Wells, High Throughput Bioanalytical Sample Preparation. Methods andAutomation Strategies, Elsevier (2003)). The amount of protein removedor separated from the test sample can be about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%. Further details regardingacridinium-9-carboxylate aryl ester and its use are set forth in U.S.patent application Ser. No. 11/697,835, filed Apr. 9, 2007.Acridinium-9-carboxylate aryl esters can be dissolved in any suitablesolvent, such as degassed anhydrous N,N-dimethylformamide (DMF) oraqueous sodium cholate.

“Linking sequence” or “linking peptide sequence” refers to a natural orartificial polypeptide sequence that is connected to one or morepolypeptide sequences of interest (e.g., full-length, fragments, etc.).The term “connected” refers to the joining of the linking sequence tothe polypeptide sequence of interest. Such polypeptide sequences arepreferably joined by one or more peptide bonds. Linking sequences canhave a length of from about 4 to about 50 amino acids. Preferably, thelength of the linking sequence is from about 6 to about 30 amino acids.Natural linking sequences can be modified by amino acid substitutions,additions, or deletions to create artificial linking sequences. Linkingsequences can be used for many purposes, including in recombinant Fabs.Exemplary linking sequences include, but are not limited to: (i)Histidine (His) tags, such as a 6×His tag, which has an amino acidsequence of HHHHHH (SEQ ID NO:4), are useful as linking sequences tofacilitate the isolation and purification of polypeptides and antibodiesof interest; (ii) Enterokinase cleavage sites, like His tags, are usedin the isolation and purification of proteins and antibodies ofinterest. Often, enterokinase cleavage sites are used together with Histags in the isolation and purification of proteins and antibodies ofinterest. Various enterokinase cleavage sites are known in the art.Examples of enterokinase cleavage sites include, but are not limited to,the amino acid sequence of DDDDK (SEQ ID NO:5) and derivatives thereof(e.g., ADDDDK (SEQ ID NO:6), etc.); (iii) Miscellaneous sequences can beused to link or connect the light and/or heavy chain variable regions ofsingle chain variable region fragments. Examples of other linkingsequences can be found in Bird et al., Science 242: 423-426 (1988);Huston et al., PNAS USA 85: 5879-5883 (1988); and McCafferty et al.,Nature 348: 552-554 (1990). Linking sequences also can be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. In the context of the present disclosure, the monoclonalantibody, for example, can contain a linking sequence, such as a Histag, an enterokinase cleavage site, or both.

“Magnetic resonance imaging” or “MRI” as used interchangeably hereinrefers to a medical imaging technique used in radiology to form picturesof the anatomy and the physiological processes of the body in bothhealth and disease. MRI is a form of medical imaging that measures theresponse of the atomic nuclei of body tissues to high-frequency radiowaves when placed in a strong magnetic field, and that produces imagesof the internal organs. MRI scanners, which is based on the science ofnuclear magnetic resonance (NMR), use strong magnetic fields, radiowaves, and field gradients to generate images of the inside of the body.

“Monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological.

“Multivalent binding protein” is used herein to refer to a bindingprotein comprising two or more antigen binding sites (also referred toherein as “antigen binding domains”). A multivalent binding protein ispreferably engineered to have three or more antigen binding sites, andis generally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein that can bind two or morerelated or unrelated targets, including a binding protein capable ofbinding two or more different epitopes of the same target molecule.

“Negative predictive value” or “NPV” as used interchangeably hereinrefers to the probability that a subject has a negative outcome giventhat they have a negative test result.

“Reference level” as used herein refers to an assay cutoff value that isused to assess diagnostic, prognostic, or therapeutic efficacy and thathas been linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.). An“absolute amount” as used herein refers to the absolute value of achange or difference between at least two assay results taken or sampledat different time points and, which similar to a reference level, hasbeen linked or is associated herein with various clinical parameters(e.g., presence of disease, stage of disease, severity of disease,progression, non-progression, or improvement of disease, etc.).“Absolute value” as used herein refers to the magnitude of a real number(such as, for example, the difference between two compared levels (suchas levels taken at a first time point and levels taken at a second timepoint)) without regard to its sign, i.e., regardless of whether it ispositive or negative.

This disclosure provides exemplary reference levels and absolute amounts(e.g., calculated by comparing reference levels at different timepoints). However, it is well-known that reference levels and absoluteamounts may vary depending on the nature of the immunoassay (e.g.,antibodies employed, reaction conditions, sample purity, etc.) and thatassays can be compared and standardized. It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific reference levels andabsolute amounts for those other immunoassays based on the descriptionprovided by this disclosure. Whereas the precise value of the referencelevel and absolute amounts may vary between assays, the findings asdescribed herein should be generally applicable and capable of beingextrapolated to other assays.

“Point-of-care device” refers to a device used to provide medicaldiagnostic testing at or near the point-of-care (namely, outside of alaboratory), at the time and place of patient care (such as in ahospital, physician's office, urgent or other medical care facility, apatient's home, a nursing home and/or a long term care and/or hospicefacility). Examples of point-of-care devices include those produced byAbbott Laboratories (Abbott Park, Ill.) (e.g., i-STAT and i-STATAlinity, Universal Biosensors (Rowville, Australia) (see US2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical LabProducts (Los Angeles, USA).

“Positive predictive value” or “PPV” as used interchangeably hereinrefers to the probability that a subject has a positive outcome giventhat they have a positive test result.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a reference level or control level (e.g.,“low”, “medium”, or “high” levels), can be used. Multiple calibrators(i.e., more than one calibrator or a varying amount of calibrator(s))can be used in conjunction to comprise a “sensitivity panel.”

A “receiver operating characteristic” curve or “ROC” curve refers to agraphical plot that illustrates the performance of a binary classifiersystem as its discrimination threshold is varied. For example, an ROCcurve can be a plot of the true positive rate against the false positiverate for the different possible cutoff points of a diagnostic test. Itis created by plotting the fraction of true positives out of thepositives (TPR=true positive rate) vs. the fraction of false positivesout of the negatives (FPR=false positive rate), at various thresholdsettings. TPR is also known as sensitivity, and FPR is one minus thespecificity or true negative rate. The ROC curve demonstrates thetradeoff between sensitivity and specificity (any increase insensitivity will be accompanied by a decrease in specificity); thecloser the curve follows the left-hand border and then the top border ofthe ROC space, the more accurate the test; the closer the curve comes tothe 45-degree diagonal of the ROC space, the less accurate the test; theslope of the tangent line at a cutoff point gives the likelihood ratio(LR) for that value of the test; and the area under the curve is ameasure of test accuracy.

“Recombinant antibody” and “recombinant antibodies” refer to antibodiesprepared by one or more steps, including cloning nucleic acid sequencesencoding all or a part of one or more monoclonal antibodies into anappropriate expression vector by recombinant techniques and subsequentlyexpressing the antibody in an appropriate host cell. The terms include,but are not limited to, recombinantly produced monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multi-specific or multi-valent structures formed fromantibody fragments, bifunctional antibodies, heteroconjugate Abs,DVD-Ig®s, and other antibodies as described in (i) herein.(Dual-variable domain immunoglobulins and methods for making them aredescribed in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

“Risk assessment,” “risk classification,” “risk identification,” or“risk stratification” of subjects (e.g., patients) as used herein refersto the evaluation of factors including biomarkers, to predict the riskof occurrence of future events including disease onset or diseaseprogression, so that treatment decisions regarding the subject may bemade on a more informed basis.

“Sample,” “test sample,” “specimen,” “biological sample”, “sample from asubject,” and “patient sample” as used herein may be usedinterchangeable and may be a sample of blood such as whole blood,tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid,placental cells or tissue, endothelial cells, leukocytes, or monocytes.The sample can be used directly as obtained from a patient or can bepre-treated, such as by filtration, distillation, extraction,concentration, centrifugation, inactivation of interfering components,addition of reagents, and the like, to modify the character of thesample in some manner as discussed herein or otherwise as is known inthe art.

A variety of cell types, tissue, or bodily fluid may be utilized toobtain a sample. Such cell types, tissues, and fluid may includesections of tissues such as biopsy and autopsy samples, frozen sectionstaken for histologic purposes, blood (such as whole blood), plasma,serum, red blood cells, platelets, interstitial fluid, cerebral spinalfluid, etc. Cell types and tissues may also include lymph fluid,cerebrospinal fluid, a fluid collected by A tissue or cell type may beprovided by removing a sample of cells from a human and a non-humananimal, but can also be accomplished by using previously isolated cells(e.g., isolated by another person, at another time, and/or for anotherpurpose). Archival tissues, such as those having treatment or outcomehistory, may also be used. Protein or nucleotide isolation and/orpurification may not be necessary.

“Sensitivity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is positive that are correctly identifiedas positive (e.g., correctly identifying those subjects with a diseaseor medical condition for which they are being tested). For example, thismight include correctly identifying subjects as having a TBI from thosewho do not have a TBI, correctly identifying subjects having a moderate,severe, or moderate to severe TBI from those having a mild TBI,correctly identifying subjects as having a mild TBI from those having amoderate, severe, or moderate to severe TBI, correctly identifyingsubjects as having a moderate, severe, or moderate to severe TBI fromthose having no TBI or correctly identifying subjects as having a mildTBI from those having no TBI, correctly identifying subjects as likelyto benefit from imaging or a head CT scan or a MRI from those who arenot likely to benefit from a head imaging or a CT scan or MRI, etc.).

“Specificity” of an assay as used herein refers to the proportion ofsubjects for whom the outcome is negative that are correctly identifiedas negative (e.g., correctly identifying those subjects who do not havea disease or medical condition for which they are being tested). Forexample, this might include correctly identifying subjects having an TBIfrom those who do not have a TBI, correctly identifying subjects nothaving a moderate, severe, or moderate to severe TBI from those having amild TBI, correctly identifying subjects as not having a mild TBI fromthose having a moderate, severe, or moderate to severe TBI or correctlyidentifying subjects as not having any TBI, or correctly identifyingsubjects as having a mild TBI from those having no TBI, etc.).

“Series of calibrating compositions” refers to a plurality ofcompositions comprising a known concentration of an analyte, such ascTnI or an early biomarker, such as UCH-L1, GFAP, or a combinationthereof, wherein each of the compositions differs from the othercompositions in the series by the concentration of the analyte.

“Solid phase” or “solid support” as used interchangeably herein, refersto any material that can be used to attach and/or attract and immobilize(1) one or more capture agents or capture specific binding partners, or(2) one or more detection agents or detection specific binding partners.The solid phase can be chosen for its intrinsic ability to attract andimmobilize a capture agent. Alternatively, the solid phase can haveaffixed thereto a linking agent that has the ability to attract andimmobilize the (1) capture agent or capture specific binding partner, or(2) detection agent or detection specific binding partner. For example,the linking agent can include a charged substance that is oppositelycharged with respect to the capture agent (e.g., capture specificbinding partner) or detection agent (e.g., detection specific bindingpartner) itself or to a charged substance conjugated to the (1) captureagent or capture specific binding partner or (2) detection agent ordetection specific binding partner. In general, the linking agent can beany binding partner (preferably specific) that is immobilized on(attached to) the solid phase and that has the ability to immobilize the(1) capture agent or capture specific binding partner, or (2) detectionagent or detection specific binding partner through a binding reaction.The linking agent enables the indirect binding of the capture agent to asolid phase material before the performance of the assay or during theperformance of the assay. For examples, the solid phase can be plastic,derivatized plastic, magnetic, or non-magnetic metal, glass or silicon,including, for example, a test tube, microtiter well, sheet, bead,microparticle, chip, and other configurations known to those of ordinaryskill in the art.

“Specific binding” or “specifically binding” as used herein may refer tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, wherein the interaction is dependent upon the presenceof a particular structure (e.g., an antigenic determinant or epitope) onthe chemical species; for example, an antibody recognizes and binds to aspecific protein structure rather than to proteins generally. If anantibody is specific for epitope “A”, the presence of a moleculecontaining epitope A (or free, unlabeled A), in a reaction containinglabeled “A” and the antibody, will reduce the amount of labeled A boundto the antibody.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzymes and enzyme inhibitors, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes and fragments thereof, whether isolatedor recombinantly produced.

“Statistically significant” as used herein refers to the likelihood thata relationship between two or more variables is caused by somethingother than random chance. Statistical hypothesis testing is used todetermine whether the result of a data set is statistically significant.In statistical hypothesis testing, a statistical significant result isattained whenever the observed p-value of a test statistic is less thanthe significance level defined of the study. The p-value is theprobability of obtaining results at least as extreme as those observed,given that the null hypothesis is true. Examples of statisticalhypothesis analysis include Wilcoxon signed-rank test, t-test,Chi-Square or Fisher's exact test. “Significant” as used herein refersto a change that has not been determined to be statistically significant(e.g., it may not have been subject to statistical hypothesis testing).

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. In someembodiments, the subject is a human. The subject or patient may beundergoing other forms of treatment. In some embodiments, when thesubject is a human, the subject does not include any humans who havesuffered a cerebrovascular accident (e.g., a stroke).

“Treat,” “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease and/or injury, or one or more symptoms of such disease, to whichsuch term applies. Depending on the condition of the subject, the termalso refers to preventing a disease, and includes preventing the onsetof a disease, or preventing the symptoms associated with a disease. Atreatment may be either performed in an acute or chronic way. The termalso refers to reducing the severity of a disease or symptoms associatedwith such disease prior to affliction with the disease. Such preventionor reduction of the severity of a disease prior to affliction refers toadministration of a pharmaceutical composition to a subject that is notat the time of administration afflicted with the disease. “Preventing”also refers to preventing the recurrence of a disease or of one or moresymptoms associated with such disease. “Treatment” and“therapeutically,” refer to the act of treating, as “treating” isdefined above.

“Traumatic Brain Injury” or “TBI” as used interchangeably herein refersto a complex injury with a broad spectrum of symptoms and disabilities.TBI is most often an acute event similar to other injuries. TBI can beclassified as “mild,” “moderate,” or “severe.” The causes of TBI arediverse and include, for example, physical shaking by a person, a caraccident, injuries from firearms, cerebral vascular accidents (e.g.,strokes), falls, explosions or blasts and other types of blunt forcetrauma. Other causes of TBI include the ingestion and/or exposure to oneor more chemicals or toxins (such as fires, molds, asbestos, pesticidesand insecticides, organic solvents, paints, glues, gases (such as carbonmonoxide, hydrogen sulfide, and cyanide), organic metals (such as methylmercury, tetraethyl lead and organic tin), one or more drugs of abuse orcombinations thereof). Alternatively, TBI can occur in subjectssuffering from an autoimmune disease, a metabolic disorder, a braintumor, hypoxia, one or more viruses, meningitis, hydrocephalus orcombinations thereof. Young adults and the elderly are the age groups athighest risk for TBI. In certain embodiments herein, traumatic braininjury or TBI does not include and specifically excludes cerebralvascular accidents such as strokes.

“Mild TBI” as used herein refers to a brain injury where loss ofconsciousness is brief and usually a few seconds or minutes and/orconfusion and disorientation is shorter than 1 hour. Mild TBI is alsoreferred to as a concussion, minor head trauma, minor TBI, minor braininjury, and minor head injury. While MRI and CT scans are often normal,the individual with mild TBI may have cognitive problems such asheadache, difficulty thinking, memory problems, attention deficits, moodswings and frustration.

Mild TBI is the most prevalent TBI and is often missed at time ofinitial injury. Typically, a subject has a Glasgow Coma scale number ofbetween 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of peoplewith mild TBI have symptoms that last 3 months or more. Mild TBI isdefined as the result of the forceful motion of the head or impactcausing a brief change in mental status (confusion, disorientation orloss of memory) or loss of consciousness for less than 30 minutes.Common symptoms of mild TBI include fatigue, headaches, visualdisturbances, memory loss, poor attention/concentration, sleepdisturbances, dizziness/loss of balance, irritability-emotionaldisturbances, feelings of depression, and seizures. Other symptomsassociated with mild TBI include nausea, loss of smell, sensitivity tolight and sounds, mood changes, getting lost or confused, and/orslowness in thinking.

“Moderate TBI” as used herein refers to a brain injury where loss ofconsciousness and/or confusion and disorientation is between 1 and 24hours and the subject has a Glasgow Coma scale number of between 9-13(such as 9-12 or 9-13). The individual with moderate TBI have abnormalbrain imaging results. “Severe TBI” as used herein refers to a braininjury where loss of consciousness is more than 24 hours and memory lossafter the injury or penetrating skull injury longer than 24 hours andthe subject has a Glasgow Coma scale number between 3-8. The deficitsrange from impairment of higher level cognitive functions to comatosestates. Survivors may have limited function of arms or legs, abnormalspeech or language, loss of thinking ability or emotional problems.Individuals with severe injuries can be left in long-term unresponsivestates. For many people with severe TBI, long-term rehabilitation isoften necessary to maximize function and independence.

“Moderate to severe” TBI as used herein refers to a spectrum of braininjury that includes moderate to severe and thus encompasses moderateTBI alone, severe TBI alone and moderate to severe TBI combined.Subjects suffering from a moderate to severe TBI can have a Glasgow Comascale number of between 3-13 (such as 3-12 or 3-13). For example, insome clinical situations, a subject may initially be diagnosed as havinga moderate TBI but who, over the course of time (minutes, hours ordays), progress to having a severe TBI (such, as for example, insituations when there is a brain bleed). Such subjects would be examplesof patients that could be classified as “moderate to severe”. Commonsymptoms of moderate to severe TBI include cognitive deficits includingdifficulties with attention, concentration, distractibility, memory,speed of processing, confusion, perseveration, impulsiveness, languageprocessing, and/or “executive functions”, not understanding the spokenword (receptive aphasia), difficulty speaking and being understood(expressive aphasia), slurred speech, speaking very fast or very slow,problems reading, problems writing, difficulties with interpretation oftouch, temperature, movement, limb position and fine discrimination, theintegration or patterning of sensory impressions into psychologicallymeaningful data, partial or total loss of vision, weakness of eyemuscles and double vision (diplopia), blurred vision, problems judgingdistance, involuntary eye movements (nystagmus), intolerance of light(photophobia), hearing, such as decrease or loss of hearing, ringing inthe ears (tinnitus), increased sensitivity to sounds, loss or diminishedsense of smell (anosmia), loss or diminished sense of taste, theconvulsions associated with epilepsy that can be several types and caninvolve disruption in consciousness, sensory perception, or motormovements, control of bowel and bladder, sleep disorders, loss ofstamina, appetite changes, regulation of body temperature, menstrualdifficulties, dependent behaviors, emotional ability, lack ofmotivation, irritability, aggression, depression, disinhibition, ordenial/lack of awareness.

“Ubiquitin carboxy-terminal hydrolase L1” or “UCH-L1” as usedinterchangeably herein refers to a deubiquitinating enzyme encoded bythe UCH-L1 gene in humans. UCH-L1, also known as ubiquitincarboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a memberof a gene family whose products hydrolyze small C-terminal adducts ofubiquitin to generate the ubiquitin monomer.

“UCH-L1 status” can mean either the level or amount of UCH-L1 at a pointin time (such as with a single measure of UCH-L1), the level or amountof UCH-L1 associated with monitoring (such as with a repeat test on asubject to identify an increase or decrease in UCH-L1 amount), the levelor amount of UCH-L1 associated with treatment for traumatic brain injury(whether a primary brain injury and/or a secondary brain injury) orcombinations thereof.

“Variant” is used herein to describe a peptide or polypeptide thatdiffers in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Representative examples of “biological activity”include the ability to be bound by a specific antibody or to promote animmune response. Variant is also used herein to describe a protein withan amino acid sequence that is substantially identical to a referencedprotein with an amino acid sequence that retains at least one biologicalactivity. A conservative substitution of an amino acid, i.e., replacingan amino acid with a different amino acid of similar properties (e.g.,hydrophilicity, degree, and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to refer to an antigenically reactivefragment of an anti-cTnI and/or anti-UCH-L1 and/or anti-GFAP antibodythat differs from the corresponding fragment of anti-cTnI and/oranti-UCH-L1 and/or anti-GFAP antibody in amino acid sequence but isstill antigenically reactive and can compete with the correspondingfragment of anti-cTnI and/or anti-UCH-L1 and/or anti-GFAP antibody forbinding with cTnI and/or anti-UCH-L1 and/or anti-GFAP. “Variant” alsocan be used to describe a polypeptide or a fragment thereof that hasbeen differentially processed, such as by proteolysis, phosphorylation,or other post-translational modification, yet retains its antigenreactivity.

“Vector” is used herein to describe a nucleic acid molecule that cantransport another nucleic acid to which it has been linked. One type ofvector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors can replicate autonomously in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”). Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. “Plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions, can be used. In this regard,RNA versions of vectors (including RNA viral vectors) may also find usein the context of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. For example,any nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those that are well known and commonly used in the art. Themeaning and scope of the terms should be clear; in the event, however ofany latent ambiguity, definitions provided herein take precedent overany dictionary or extrinsic definition. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

2. METHODS OF AIDING IN THE DIAGNOSIS AND EVALUATION OF WHETHER A HUMANSUBJECT MAY HAVE SUSTAINED OR HAS SUSTAINED AN (HAS AN ACTUAL ORSUSPECTED) INJURY TO THE HEAD USING CARDIAC TROPONIN I (CTNI) AND ANEARLY BIOMARKER

The present disclosure relates, among other methods, to a method ofaiding in the diagnosis and evaluation of whether a human subject hassustained or may have sustained an (or has an actual or suspected)injury to the head. The method can aid in determining the extent oftraumatic brain injury in a human subject with an actual suspectedinjury to the head, e.g., determining whether the subject has mildtraumatic brain injury or a moderate, severe, or moderate to severetraumatic brain injury. As used here, “determining whether the subjecthas mild traumatic brain injury or a moderate, severe, or moderate tosevere” refers to the fact that the aforementioned method can be used,e.g., with other information (e.g., clinical assessment data), todetermine that the subject is more likely than not to have mildtraumatic brain injury or moderate to severe traumatic brain injury. Themethod can include performing an assay on a sample obtained from thehuman subject within about 24 hours, such as within about 2 hours, afteran actual or suspected injury to the head to measure or detect a levelof cardiac troponin I (cTnI) and a level of an early biomarker in thesample and determining whether the subject has sustained a mild or amoderate, severe, or moderate to severe traumatic brain injury (TBI). Insome embodiments, the subject is determined as having (1) a moderate,severe, or a moderate to severe TBI when the level of cTnI in the sampleis higher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker, or (2) a mild TBI when the level of cTnI in the sample islower than a reference level of cTnI and/or the level of the earlybiomarker in the sample is lower than a reference level of the earlybiomarker. The sample can be a biological sample. The early biomarkerincludes ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glialfibrillary acidic protein (GFAP), or a combination thereof.

In some embodiments, the method can include obtaining a sample withinabout 24 hours, such as within about 2 hours, of an actual or suspectedinjury to the subject and contacting the sample with an antibody forcTnI to allow formation of a complex of the antibody and cTnI and withan antibody for the early biomarker to allow formation of a complex ofthe antibody and the early biomarker. The method also includes detectingthe resulting antibody-cTnI complex and the resulting antibody-earlybiomarker complex.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of an actual or suspect injury to the head.

In some embodiments, the sample is taken from the human subject withinabout 2 hours of injury or suspected injury to the head. For example,the sample can be taken from the human subject within about 0 minutes,about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 60 minutes, about 90 minutes, or about 2 hours of injuryor suspected injury to the head. In some embodiments, the onset of thepresence of cTnI and/or the early biomarker appears within about 0minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about2 hours after injury to the head.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cTnI and/or the early biomarker isdetermined at one or more time points. In certain embodiments, thesubject may be suspected of having a mild traumatic brain injury basedon the Glasgow Coma Scale score. In certain embodiments, the subject maybe suspected of having a mild traumatic brain injury based on anabnormal head CT. In some embodiments, the subject has received a CTscan before or after the assay is performed. In some embodiments, thesubject has a normal head CT.

In some embodiments, the reference level of cTnI and the reference levelof the early biomarker are correlated with a Glasgow Coma Scale score of3-12 (moderate to severe TBI). In some embodiments, the reference levelof cTnI and the reference level of the early biomarker are correlatedwith a Glasgow Coma Scale score of 3-8 (a severe TBI). In someembodiments, the reference level of cTnI and the reference level of theearly biomarker are correlated with a Glasgow Coma Scale score of 9-13(a moderate TBI). In some embodiments, the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore. In some embodiments, the reference level of cTnI and thereference level of the early biomarker are correlated with subjectshaving mild traumatic brain injury. In some embodiments, the referencelevel of cTnI and the reference level of the early biomarker arecorrelated with a Glasgow Coma Scale score of 13-15 (a mild TBI).

Generally, a reference level of cTnI and a reference level of an earlybiomarker can also be employed as a benchmark against which to assessresults obtained upon assaying a test sample for cTnI and earlybiomarker, respectively. Generally, in making such a comparison, thereference level of cTnI and the reference level of the early biomarkerare obtained by running a particular assay a sufficient number of timesand under appropriate conditions such that a linkage or association ofanalyte presence, amount or concentration with a particular stage orendpoint of TBI or with particular indicia can be made. Typically, thereference level of cTnI and the reference level of the early biomarkerare obtained with assays of reference subjects (or populations ofsubjects). The cTnI and/or early biomarker measured can includefragments thereof, degradation products thereof, and/or enzymaticcleavage products thereof.

In certain embodiments, the reference level may be correlated withcontrol subjects that have not sustained a head injury.

In some embodiments, the reference level of cTnI and/or reference levelof the early biomarker is determined by an assay having a sensitivity ofbetween at least about 65% to about 100% and a specificity of between atleast about 30% to about 100%. In some embodiments, the sensitivity isbetween at least about 65% to about 100%, between at least about 65% toat least about 99%, between at least about 65% to at least about 95%,between at least about 65% to at least about 90%, between at least about65% to at least about 85%, between at least about 65% to at least about80%, between at least about 65% to at least about 75%, between at leastabout 65% to at least about 70%, between at least about 75% to about100%, between at least about 75% to at least about 99%, between at leastabout 75% to at least about 95%, between at least about 75% to at leastabout 90%, between at least about 75% to at least about 85%, between atleast about 75% to at least about 80%, between at least about 85% toabout 100%, between at least about 85% to at least about 99%, between atleast about 85% to at least about 95%, between at least about 85% to atleast about 90%, between at least about 95% to about 100%, or between atleast about 95% to at least about 99%. In some embodiments, thesensitivity is at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%,at least about 99.1%, at least about 99.2%, at least about 99.3%, atleast about 99.4%, at least about 99.5%, at least about 99.6%, at leastabout 99.7%, at least about 99.8%, at least about 99.9%, or at leastabout 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the amount of cTnI in the sample is from about 1pg/mL to about 100 pg/mL, about 1 pg/mL to about 90 pg/mL, about 1 pg/mLto about 80 pg/mL, about 1 pg/mL to about 70 pg/mL, about 1 pg/mL toabout 60 pg/mL, about 1 pg/mL to about 55 pg/mL, about 1 pg/mL to about50 pg/mL, about 1 pg/mL to about 45 pg/mL, about 1 pg/mL to about 40pg/mL, about 1 pg/mL to about 35 pg/mL, about 1 pg/mL to about 30 pg/mL,about 1 pg/mL to about 25 pg/mL, about 1 pg/mL to about 20 pg/mL, about1 pg/mL to about 15 pg/mL, about 1 pg/mL to about 10 pg/mL, about 1pg/mL to about 9 pg/mL, about 1 pg/mL to about 8 pg/mL, about 1 pg/mL toabout 7 pg/mL, about 1 pg/mL to about 6 pg/mL, about 1 pg/mL to about 5pg/mL, about 1 pg/mL to about 4 pg/mL, about 1 pg/mL to about 3 pg/mL,about 1 pg/mL to about 2 pg/mL, about 1 pg/mL to about 1.5 pg/mL, about1.5 pg/mL to about 100 pg/mL, about 1.5 pg/mL to about 90 pg/mL, about1.5 pg/mL to about 80 pg/mL, about 1.5 pg/mL to about 70 pg/mL, about1.5 pg/mL to about 60 pg/mL, about 1.5 pg/mL to about 55 pg/mL, about1.5 pg/mL to about 50 pg/mL, about 1.5 pg/mL to about 45 pg/mL, about1.5 pg/mL to about 40 pg/mL, about 1.5 pg/mL to about 35 pg/mL, about1.5 pg/mL to about 30 pg/mL, about 1.5 pg/mL to about 25 pg/mL, about1.5 pg/mL to about 20 pg/mL, about 1.5 pg/mL to about 15 pg/mL, about1.5 pg/mL to about 10 pg/mL, about 1.5 pg/mL to about 9 pg/mL, about 1.5pg/mL to about 8 pg/mL, about 1.5 pg/mL to about 7 pg/mL, about 1.5pg/mL to about 6 pg/mL, about 1.5 pg/mL to about 5 pg/mL, about 1.5pg/mL to about 4 pg/mL, about 1.5 pg/mL to about 3 pg/mL, about 1.5pg/mL to about 2 pg/mL, about 2 pg/mL to about 100 pg/mL, about 2 pg/mLto about 90 pg/mL, about 2 pg/mL to about 80 pg/mL, about 2 pg/mL toabout 70 pg/mL, about 2 pg/mL to about 60 pg/mL, about 2 pg/mL to about55 pg/mL, about 2 pg/mL to about 50 pg/mL, about 2 pg/mL to about 45pg/mL, about 2 pg/mL to about 40 pg/mL, about 2 pg/mL to about 35 pg/mL,about 2 pg/mL to about 30 pg/mL, about 2 pg/mL to about 25 pg/mL, about2 pg/mL to about 20 pg/mL, about 2 pg/mL to about 15 pg/mL, about 2pg/mL to about 10 pg/mL, about 2 pg/mL to about 9 pg/mL, about 2 pg/mLto about 8 pg/mL, about 2 pg/mL to about 7 pg/mL, about 2 pg/mL to about6 pg/mL, about 2 pg/mL to about 5 pg/mL, about 2 pg/mL to about 4 pg/mL,about 2 pg/mL to about 3 pg/mL, about 3 pg/mL to about 100 pg/mL, about3 pg/mL to about 90 pg/mL, about 3 pg/mL to about 80 pg/mL, about 3pg/mL to about 70 pg/mL, about 3 pg/mL to about 60 pg/mL, about 3 pg/mLto about 55 pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL toabout 45 pg/mL, about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about35 pg/mL, about 3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25pg/mL, about 3 pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL,about 3 pg/mL to about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3pg/mL to about 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL toabout 6 pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4pg/mL, about 4 pg/mL to about 100 pg/mL, about 4 pg/mL to about 90pg/mL, about 4 pg/mL to about 80 pg/mL, about 4 pg/mL to about 70 pg/mL,about 4 pg/mL to about 60 pg/mL, about 4 pg/mL to about 55 pg/mL, about4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about 4pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4 pg/mLto about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mL toabout 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL to about10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 100 pg/mL, about5 pg/mL to about 90 pg/mL, about 5 pg/mL to about 80 pg/mL, about 5pg/mL to about 70 pg/mL, about 5 pg/mL to about 60 pg/mL, about 5 pg/mLto about 55 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5 pg/mL toabout 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mL to about35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL to about 25pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about 15 pg/mL,about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9 pg/mL, about 5pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL, about 5 pg/mL toabout 6 pg/mL, about 6 pg/mL to about 100 pg/mL, about 6 pg/mL to about90 pg/mL, about 6 pg/mL to about 80 pg/mL, about 6 pg/mL to about 70pg/mL, about 6 pg/mL to about 60 pg/mL, about 6 pg/mL to about 55 pg/mL,about 6 pg/mL to about 50 pg/mL, about 6 pg/mL to about 45 pg/mL, about6 pg/mL to about 40 pg/mL, about 6 pg/mL to about 35 pg/mL, about 6pg/mL to about 30 pg/mL, about 6 pg/mL to about 25 pg/mL, about 6 pg/mLto about 20 pg/mL, about 6 pg/mL to about 15 pg/mL, about 6 pg/mL toabout 10 pg/mL, about 6 pg/mL to about 9 pg/mL, about 6 pg/mL to about 8pg/mL, about 6 pg/mL to about 7 pg/mL, about 7 pg/mL to about 100 pg/mL,about 7 pg/mL to about 90 pg/mL, about 7 pg/mL to about 80 pg/mL, about7 pg/mL to about 70 pg/mL, about 7 pg/mL to about 60 pg/mL, about 7pg/mL to about 55 pg/mL, about 7 pg/mL to about 50 pg/mL, about 7 pg/mLto about 45 pg/mL, about 7 pg/mL to about 40 pg/mL, about 7 pg/mL toabout 35 pg/mL, about 7 pg/mL to about 30 pg/mL, about 7 pg/mL to about25 pg/mL, about 7 pg/mL to about 20 pg/mL, about 7 pg/mL to about 15pg/mL, about 7 pg/mL to about 10 pg/mL, about 7 pg/mL to about 9 pg/mL,about 7 pg/mL to about 8 pg/mL, about 8 pg/mL to about 100 pg/mL, about8 pg/mL to about 90 pg/mL, about 8 pg/mL to about 80 pg/mL, about 8pg/mL to about 70 pg/mL, about 8 pg/mL to about 60 pg/mL, about 8 pg/mLto about 55 pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL toabout 45 pg/mL, about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about35 pg/mL, about 8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25pg/mL, about 8 pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL,about 8 pg/mL to about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9pg/mL to about 100 pg/mL, about 9 pg/mL to about 90 pg/mL, about 9 pg/mLto about 80 pg/mL, about 9 pg/mL to about 70 pg/mL, about 9 pg/mL toabout 60 pg/mL, about 9 pg/mL to about 55 pg/mL, about 9 pg/mL to about50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about 40pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30 pg/mL,about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL, about9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about 10pg/mL to about 100 pg/mL, about 10 pg/mL to about 90 pg/mL, about 10pg/mL to about 80 pg/mL, about 10 pg/mL to about 70 pg/mL, about 10pg/mL to about 60 pg/mL, about 10 pg/mL to about 55 pg/mL, about 10pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 100 pg/mL, about 20 pg/mL to about 90 pg/mL, about 20pg/mL to about 80 pg/mL, about 20 pg/mL to about 70 pg/mL, about 20pg/mL to about 60 pg/mL, about 20 pg/mL to about 55 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the amount of cTnI can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, atleast about 50 pg/mL, at least about 60 pg/mL, at least about 70 pg/mL,at least about 80 pg/mL, at least about 90 pg/mL, or at least about 100pg/mL.

In some embodiments, the amount of the early biomarker, such as UCH-L1,GFAP, or a combination thereof, can be between at least about 1 pg/mL toabout 1000 pg/mL. In some embodiments, the reference level of the earlybiomarker, such as UCH-L1, GFAP, or a combination thereof, can bebetween at least about 1 pg/mL to about 1000 pg/mL, between at leastabout 1 pg/mL to about 900 pg/mL, between at least about 1 pg/mL toabout 800 pg/mL, between at least about 1 pg/mL to about 700 pg/mL,between at least about 1 pg/mL to about 600 pg/mL, between at leastabout 1 pg/mL to about 550 pg/mL, between at least about 1 pg/mL toabout 500 pg/mL, between at least about 1 pg/mL to about 450 pg/mL,between at least about 1 pg/mL to about 400 pg/mL, between at leastabout 1 pg/mL to about 300 pg/mL, between at least about 1 pg/mL toabout 300 pg/mL, between at least about 1 pg/mL to about 200 pg/mL,between at least about 1 pg/mL to about 100 pg/mL, between at leastabout 1 pg/mL to about 50 pg/mL, between at least about 1 pg/mL to about20 pg/mL, between at least about 1 pg/mL to about 15 pg/mL, between atleast about 1 pg/mL to about 10 pg/mL, between at least about 5 pg/mL toabout 1000 pg/mL, between at least about 5 pg/mL to about 900 pg/mL,between at least about 5 pg/mL to about 800 pg/mL, between at leastabout 5 pg/mL to about 700 pg/mL, between at least about 5 pg/mL toabout 600 pg/mL, between at least about 5 pg/mL to about 550 pg/mL,between at least about 5 pg/mL to about 500 pg/mL, between at leastabout 5 pg/mL to about 450 pg/mL, between at least about 5 pg/mL toabout 400 pg/mL, between at least about 5 pg/mL to about 300 pg/mL,between at least about 5 pg/mL to about 300 pg/mL, between at leastabout 5 pg/mL to about 200 pg/mL, between at least about 5 pg/mL toabout 100 pg/mL, between at least about 5 pg/mL to about 50 pg/mL,between at least about 5 pg/mL to about 20 pg/mL, between at least about5 pg/mL to about 15 pg/mL, between at least about 5 pg/mL to about 10pg/mL, between at least about 10 pg/mL to about 1000 pg/mL, between atleast about 10 pg/mL to about 900 pg/mL, between at least about 10 pg/mLto about 800 pg/mL, between at least about 10 pg/mL to about 700 pg/mL,between at least about 10 pg/mL to about 600 pg/mL, between at leastabout 10 pg/mL to about 550 pg/mL, between at least about 10 pg/mL toabout 500 pg/mL, between at least about 10 pg/mL to about 450 pg/mL,between at least about 10 pg/mL to about 400 pg/mL, between at leastabout 10 pg/mL to about 300 pg/mL, between at least about 10 pg/mL toabout 200 pg/mL, between at least about 10 pg/mL to about 100 pg/mL,between at least about 10 pg/mL to about 50 pg/mL, between at leastabout 10 pg/mL to about 20 pg/mL, between at least about 10 pg/mL toabout 15 pg/mL, between at least about 25 pg/mL to about 1000 pg/mL,between at least about 25 pg/mL to about 900 pg/mL, between at leastabout 25 pg/mL to about 800 pg/mL, between at least about 25 pg/mL toabout 700 pg/mL, between at least about 25 pg/mL to about 600 pg/mL,between at least about 25 pg/mL to about 525 pg/mL, between at leastabout 25 pg/mL to about 500 pg/mL, between at least about 25 pg/mL toabout 425 pg/mL, between at least about 25 pg/mL to about 400 pg/mL,between at least about 25 pg/mL to about 300 pg/mL, between at leastabout 25 pg/mL to about 200 pg/mL, between at least about 25 pg/mL toabout 100 pg/mL, between at least about 25 pg/mL to about 50 pg/mL,between at least about 50 pg/mL to about 1000 pg/mL, between at leastabout 50 pg/mL to about 900 pg/mL, between at least about 50 pg/mL toabout 800 pg/mL, between at least about 50 pg/mL to about 700 pg/mL,between at least about 50 pg/mL to about 600 pg/mL, between at leastabout 50 pg/mL to about 550 pg/mL, between at least about 50 pg/mL toabout 500 pg/mL, between at least about 50 pg/mL to about 450 pg/mL,between at least about 50 pg/mL to about 400 pg/mL, between at leastabout 50 pg/mL to about 300 pg/mL, between at least about 50 pg/mL toabout 200 pg/mL, between at least about 50 pg/mL to about 100 pg/mL,between at least about 100 pg/mL to about 1000 pg/mL, between at leastabout 100 pg/mL to about 900 pg/mL, between at least about 100 pg/mL toabout 800 pg/mL, between at least about 100 pg/mL to about 700 pg/mL,between at least about 100 pg/mL to about 600 pg/mL, between at leastabout 100 pg/mL to about 550 pg/mL, between at least about 100 pg/mL toabout 500 pg/mL, between at least about 100 pg/mL to about 450 pg/mL,between at least about 100 pg/mL to about 400 pg/mL, between at leastabout 100 pg/mL to about 300 pg/mL, between at least about 100 pg/mL toabout 200 pg/mL, between at least about 200 pg/mL to about 1000 pg/mL,between at least about 200 pg/mL to about 900 pg/mL, between at leastabout 200 pg/mL to about 800 pg/mL, between at least about 200 pg/mL toabout 700 pg/mL, between at least about 200 pg/mL to about 600 pg/mL,between at least about 200 pg/mL to about 550 pg/mL, between at leastabout 200 pg/mL to about 500 pg/mL, between at least about 200 pg/mL toabout 450 pg/mL, between at least about 200 pg/mL to about 400 pg/mL,between at least about 200 pg/mL to about 300 pg/mL, between at leastabout 300 pg/mL to about 1000 pg/mL, between at least about 300 pg/mL toabout 900 pg/mL, between at least about 300 pg/mL to about 800 pg/mL,between at least about 300 pg/mL to about 700 pg/mL, between at leastabout 300 pg/mL to about 600 pg/mL, between at least about 300 pg/mL toabout 550 pg/mL, between at least about 300 pg/mL to about 500 pg/mL,between at least about 300 pg/mL to about 450 pg/mL, between at leastabout 300 pg/mL to about 400 pg/mL, between at least about 400 pg/mL toabout 1000 pg/mL, between at least about 400 pg/mL to about 900 pg/mL,between at least about 400 pg/mL to about 800 pg/mL, between at leastabout 400 pg/mL to about 700 pg/mL, between at least about 400 pg/mL toabout 600 pg/mL, between at least about 400 pg/mL to about 550 pg/mL,between at least about 400 pg/mL to about 500 pg/mL, between at leastabout 400 pg/mL to about 450 pg/mL, between at least about 500 pg/mL toabout 1000 pg/mL, between at least about 500 pg/mL to about 900 pg/mL,between at least about 500 pg/mL to about 800 pg/mL, between at leastabout 500 pg/mL to about 700 pg/mL, between at least about 500 pg/mL toabout 600 pg/mL, between at least about 500 pg/mL to about 550 pg/mL,between at least about 600 pg/mL to about 1000 pg/mL, between at leastabout 600 pg/mL to about 900 pg/mL, between at least about 600 pg/mL toabout 800 pg/mL, or between at least about 600 pg/mL to about 700 pg/mL.In some embodiments, the amount of the early biomarker, such as UCH-L1,GFAP, or a combination thereof, can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, atleast about 50 pg/mL, at least about 100 pg/mL, at least about 150pg/mL, at least about 200 pg/mL, at least about 250 pg/mL, at leastabout 300 pg/mL, at least about 350 pg/mL, at least about 400 pg/mL, atleast about 450 pg/mL, at least about 500 pg/mL, at least about 550pg/mL, at least about 600 pg/mL, at least about 650 pg/mL, at leastabout 700 pg/mL, at least about 750 pg/mL, at least about 800 pg/mL, atleast about 850 pg/mL, at least about 900 pg/mL, at least about 950pg/mL, or at least about 1000 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury with a traumatic brain injury treatment, asdescribed below. In some embodiments, the method further includesmonitoring the human subject assessed as having mild traumatic braininjury, as described below. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blotanalysis, or protein immunostaining, electrophoresis analysis, a proteinassay, a competitive binding assay, a functional protein assay, orchromatography or spectrometry methods, such as high-performance liquidchromatography (HPLC) or liquid chromatography-mass spectrometry(LC/MS). Also, the assay can be employed in a clinical chemistry formatsuch as would be known by one of ordinary skill in the art. Such assaysare described in further detail herein in Sections 8-12. It is known inthe art that the values (e.g., reference levels, cutoffs, thresholds,specificities, sensitivities, concentrations of calibrators and/orcontrols etc.) used in an assay that employs specific sample type (e.g.,such as an immunoassay that utilizes serum or a point-of-care devicethat employs whole blood) can be extrapolated to other assay formatsusing known techniques in the art, such as assay standardization. Forexample, one way in which assay standardization can be performed is byapplying a factor to the calibrator employed in the assay to make thesample concentration read higher or lower to get a slope that alignswith the comparator method. Other methods of standardizing resultsobtained on one assay to another assay are well known and have beendescribed in the literature (See, for example, David Wild, ImmunoassayHandbook, 4^(th) edition, chapter 3.5, pages 315-322, the contents ofwhich are herein incorporated by reference).

3. METHOD OF AIDING IN THE DETERMINATION OF WHETHER TO PERFORM A CT SCANON A HUMAN SUBJECT WHO MAY HAVE OR HAS SUSTAINED AN (OR HAS AN ACTUAL ORSUSPECTED) INJURY TO THE HEAD USING CARDIAC TROPONIN I (CTNI) AND ANEARLY BIOMARKER

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform a computerized tomography (CT)scan on a human subject who has sustained or may have sustained an (orhas an actual or suspected) injury to the head. As used here,“determination of whether to perform a CT scan on a human subject”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have a positive head CTscan. Specifically, such a method can comprise the steps of: (a)performing an assay on a sample obtained from the subject within about24 hours, such as within about 2 hours, after an actual or suspectedinjury to the head to measure or detect a level of cardiac troponin I(cTnI) and a level of an early biomarker in the sample and the level ofthe early biomarker in the sample is higher than a reference level ofthe early biomarker; and (b) performing a CT scan on the subject whenthe level of cTnI in the sample is higher than a reference level of cTnIand not performing a CT scan on the subject when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker. The sample can be a biological sample. The earlybiomarker includes ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof.

In some embodiments, the method can include obtaining a sample withinabout 24 hours, such as within about 2 hours, of an actual suspectedinjury to the subject and contacting the sample with an antibody forcTnI to allow formation of a complex of the antibody and cTnI and withan antibody for the early biomarker to allow formation of a complex ofthe antibody and the early biomarker. The method also includes detectingthe resulting antibody-cTnI complex and the resulting antibody-earlybiomarker complex.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of a suspect injury to the head.

In some embodiments, the sample is taken from the human subject withinabout 2 hours of injury or suspected injury to the head. For example,the sample can be taken from the human subject within about 0 minutes,about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30minutes, about 60 minutes, about 90 minutes, or about 2 hours of injuryor suspected injury to the head. In some embodiments, the onset of thepresence of cTnI and/or the early biomarker appears within about 0minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about2 hours after injury to the head.

In some embodiments, the subject has received a CT scan before or afterthe assay is performed. In some embodiments, the subject is suspected ashaving a traumatic brain injury based on the CT scan. In someembodiments, the reference level of cTnI and the reference level of theearly biomarker are correlated with positive head CT scan.

Generally, a reference level of cTnI and the reference level of theearly biomarker can be employed as a benchmark against which to assessresults obtained upon assaying a test sample for cTnI and earlybiomarker, respectively. Generally, in making such a comparison, thereference level of cTnI and the reference level of the early biomarkerare obtained by running a particular assay a sufficient number of timesand under appropriate conditions such that a linkage or association ofanalyte presence, amount or concentration with a particular stage orendpoint of TBI or with particular indicia can be made. Typically, thereference level of cTnI and the reference level of the early biomarkerare obtained with assays of reference subjects (or populations ofsubjects). The cTnI and/or early biomarker measured can includefragments thereof, degradation products thereof, and/or enzymaticcleavage products thereof.

In some embodiments, the reference level of cTnI and/or reference levelof the early biomarker is determined by an assay having a sensitivity ofbetween at least about 65% to about 100% and a specificity of between atleast about 30% to about 100%. In some embodiments, the sensitivity isbetween at least about 65% to about 100%, between at least about 65% toat least about 99%, between at least about 65% to at least about 95%,between at least about 65% to at least about 90%, between at least about65% to at least about 85%, between at least about 65% to at least about80%, between at least about 65% to at least about 75%, between at leastabout 65% to at least about 70%, between at least about 75% to about100%, between at least about 75% to at least about 99%, between at leastabout 75% to at least about 95%, between at least about 75% to at leastabout 90%, between at least about 75% to at least about 85%, between atleast about 75% to at least about 80%, between at least about 85% toabout 100%, between at least about 85% to at least about 99%, between atleast about 85% to at least about 95%, between at least about 85% to atleast about 90%, between at least about 95% to about 100%, or between atleast about 95% to at least about 99%. In some embodiments, thesensitivity is at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%,at least about 99.1%, at least about 99.2%, at least about 99.3%, atleast about 99.4%, at least about 99.5%, at least about 99.6%, at leastabout 99.7%, at least about 99.8%, at least about 99.9%, or at leastabout 100.0%.

In some embodiments, the specificity is between at least about 30% toabout 100%, between at least about 30% to about 99%, between at leastabout 30% to about 95%, between at least about 30% to about 90%, betweenat least about 30% to about 85%, between at least about 30% to about80%, between at least about 30% to about 75%, between at least about 30%to about 70%, between at least about 30% to about 60%, between at leastabout 30% to about 50%, between at least about 40% to about 100%,between at least about 40% to about 99%, between at least about 40% toabout 95%, between at least about 40% to about 90%, between at leastabout 40% to about 85%, between at least about 40% to about 80%, betweenat least about 40% to about 75%, between at least about 40% to about70%, between at least about 40% to about 60%, between at least about 40%to about 50%, between at least about 50% to about 100%, between at leastabout 50% to about 99%, between at least about 50% to about 95%, betweenat least about 50% to about 90%, between at least about 50% to about85%, between at least about 50% to about 80%, between at least about 50%to about 75%, between at least about 50% to about 70%, between at leastabout 50% to about 60%, between at least about 60% to about 100%,between at least about 60% to about 99%, between at least about 60% toabout 95%, between at least about 60% to about 90%, between at leastabout 60% to about 85%, between at least about 60% to about 80%, betweenat least about 60% to about 75%, between at least about 60% to about70%, between at least about 70% to about 100%, between at least about70% to about 99%, between at least about 70% to about 95%, between atleast about 70% to about 90%, between at least about 70% to about 85%,between at least about 70% to about 80%, between at least about 70% toabout 75%, between at least about 80% to about 100%, between at leastabout 80% to about 99%, between at least about 80% to about 95%, betweenat least about 80% to about 90%, between at least about 80% to about85%, between at least about 90% to about 100%, between at least about90% to about 99%, between at least about 90% to about 95%, between atleast about 95% to about 99%, or between at least about 95% to about100. In some embodiments, the specificity is at least about 30.0%, atleast about 31.0%, at least about 32.0%, at least about 33.0%, at leastabout 34.0%, at least about 35.0%, at least about 36.0%, at least about37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%,at least about 45.0%, at least about 50.0%, at least about 55.0%, atleast about 60.0%, at least about 65.0%, at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%,at least about 94.0%, at least about 95.0%, at least about 96.0%, atleast about 97.0%, at least about 98.0%, at least about 99.0%, at leastabout 99.1%, at least about 99.2%, at least about 99.3%, at least about99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%,at least about 99.8%, at least about 99.9%, or at least about 100.0%.For example, the sensitivity is at least about 99% and the specificityis at least about 75%, the sensitivity is at least about 99% and thespecificity is at least about 99%, or the sensitivity is at least about100% and the specificity is at least about 100%.

In some embodiments, the amount of cTnI in the sample is from about 1pg/mL to about 100 pg/mL, about 1 pg/mL to about 90 pg/mL, about 1 pg/mLto about 80 pg/mL, about 1 pg/mL to about 70 pg/mL, about 1 pg/mL toabout 60 pg/mL, about 1 pg/mL to about 55 pg/mL, about 1 pg/mL to about50 pg/mL, about 1 pg/mL to about 45 pg/mL, about 1 pg/mL to about 40pg/mL, about 1 pg/mL to about 35 pg/mL, about 1 pg/mL to about 30 pg/mL,about 1 pg/mL to about 25 pg/mL, about 1 pg/mL to about 20 pg/mL, about1 pg/mL to about 15 pg/mL, about 1 pg/mL to about 10 pg/mL, about 1pg/mL to about 9 pg/mL, about 1 pg/mL to about 8 pg/mL, about 1 pg/mL toabout 7 pg/mL, about 1 pg/mL to about 6 pg/mL, about 1 pg/mL to about 5pg/mL, about 1 pg/mL to about 4 pg/mL, about 1 pg/mL to about 3 pg/mL,about 1 pg/mL to about 2 pg/mL, about 1 pg/mL to about 1.5 pg/mL, about1.5 pg/mL to about 100 pg/mL, about 1.5 pg/mL to about 90 pg/mL, about1.5 pg/mL to about 80 pg/mL, about 1.5 pg/mL to about 70 pg/mL, about1.5 pg/mL to about 60 pg/mL, about 1.5 pg/mL to about 55 pg/mL, about1.5 pg/mL to about 50 pg/mL, about 1.5 pg/mL to about 45 pg/mL, about1.5 pg/mL to about 40 pg/mL, about 1.5 pg/mL to about 35 pg/mL, about1.5 pg/mL to about 30 pg/mL, about 1.5 pg/mL to about 25 pg/mL, about1.5 pg/mL to about 20 pg/mL, about 1.5 pg/mL to about 15 pg/mL, about1.5 pg/mL to about 10 pg/mL, about 1.5 pg/mL to about 9 pg/mL, about 1.5pg/mL to about 8 pg/mL, about 1.5 pg/mL to about 7 pg/mL, about 1.5pg/mL to about 6 pg/mL, about 1.5 pg/mL to about 5 pg/mL, about 1.5pg/mL to about 4 pg/mL, about 1.5 pg/mL to about 3 pg/mL, about 1.5pg/mL to about 2 pg/mL, about 2 pg/mL to about 100 pg/mL, about 2 pg/mLto about 90 pg/mL, about 2 pg/mL to about 80 pg/mL, about 2 pg/mL toabout 70 pg/mL, about 2 pg/mL to about 60 pg/mL, about 2 pg/mL to about55 pg/mL, about 2 pg/mL to about 50 pg/mL, about 2 pg/mL to about 45pg/mL, about 2 pg/mL to about 40 pg/mL, about 2 pg/mL to about 35 pg/mL,about 2 pg/mL to about 30 pg/mL, about 2 pg/mL to about 25 pg/mL, about2 pg/mL to about 20 pg/mL, about 2 pg/mL to about 15 pg/mL, about 2pg/mL to about 10 pg/mL, about 2 pg/mL to about 9 pg/mL, about 2 pg/mLto about 8 pg/mL, about 2 pg/mL to about 7 pg/mL, about 2 pg/mL to about6 pg/mL, about 2 pg/mL to about 5 pg/mL, about 2 pg/mL to about 4 pg/mL,about 2 pg/mL to about 3 pg/mL, about 3 pg/mL to about 100 pg/mL, about3 pg/mL to about 90 pg/mL, about 3 pg/mL to about 80 pg/mL, about 3pg/mL to about 70 pg/mL, about 3 pg/mL to about 60 pg/mL, about 3 pg/mLto about 55 pg/mL, about 3 pg/mL to about 50 pg/mL, about 3 pg/mL toabout 45 pg/mL, about 3 pg/mL to about 40 pg/mL, about 3 pg/mL to about35 pg/mL, about 3 pg/mL to about 30 pg/mL, about 3 pg/mL to about 25pg/mL, about 3 pg/mL to about 20 pg/mL, about 3 pg/mL to about 15 pg/mL,about 3 pg/mL to about 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3pg/mL to about 8 pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL toabout 6 pg/mL, about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4pg/mL, about 4 pg/mL to about 100 pg/mL, about 4 pg/mL to about 90pg/mL, about 4 pg/mL to about 80 pg/mL, about 4 pg/mL to about 70 pg/mL,about 4 pg/mL to about 60 pg/mL, about 4 pg/mL to about 55 pg/mL, about4 pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about 4pg/mL to about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4 pg/mLto about 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mL toabout 20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL to about10 pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8pg/mL, about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL,about 4 pg/mL to about 5 pg/mL, about 5 pg/mL to about 100 pg/mL, about5 pg/mL to about 90 pg/mL, about 5 pg/mL to about 80 pg/mL, about 5pg/mL to about 70 pg/mL, about 5 pg/mL to about 60 pg/mL, about 5 pg/mLto about 55 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5 pg/mL toabout 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mL to about35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL to about 25pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about 15 pg/mL,about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9 pg/mL, about 5pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL, about 5 pg/mL toabout 6 pg/mL, about 6 pg/mL to about 100 pg/mL, about 6 pg/mL to about90 pg/mL, about 6 pg/mL to about 80 pg/mL, about 6 pg/mL to about 70pg/mL, about 6 pg/mL to about 60 pg/mL, about 6 pg/mL to about 55 pg/mL,about 6 pg/mL to about 50 pg/mL, about 6 pg/mL to about 45 pg/mL, about6 pg/mL to about 40 pg/mL, about 6 pg/mL to about 35 pg/mL, about 6pg/mL to about 30 pg/mL, about 6 pg/mL to about 25 pg/mL, about 6 pg/mLto about 20 pg/mL, about 6 pg/mL to about 15 pg/mL, about 6 pg/mL toabout 10 pg/mL, about 6 pg/mL to about 9 pg/mL, about 6 pg/mL to about 8pg/mL, about 6 pg/mL to about 7 pg/mL, about 7 pg/mL to about 100 pg/mL,about 7 pg/mL to about 90 pg/mL, about 7 pg/mL to about 80 pg/mL, about7 pg/mL to about 70 pg/mL, about 7 pg/mL to about 60 pg/mL, about 7pg/mL to about 55 pg/mL, about 7 pg/mL to about 50 pg/mL, about 7 pg/mLto about 45 pg/mL, about 7 pg/mL to about 40 pg/mL, about 7 pg/mL toabout 35 pg/mL, about 7 pg/mL to about 30 pg/mL, about 7 pg/mL to about25 pg/mL, about 7 pg/mL to about 20 pg/mL, about 7 pg/mL to about 15pg/mL, about 7 pg/mL to about 10 pg/mL, about 7 pg/mL to about 9 pg/mL,about 7 pg/mL to about 8 pg/mL, about 8 pg/mL to about 100 pg/mL, about8 pg/mL to about 90 pg/mL, about 8 pg/mL to about 80 pg/mL, about 8pg/mL to about 70 pg/mL, about 8 pg/mL to about 60 pg/mL, about 8 pg/mLto about 55 pg/mL, about 8 pg/mL to about 50 pg/mL, about 8 pg/mL toabout 45 pg/mL, about 8 pg/mL to about 40 pg/mL, about 8 pg/mL to about35 pg/mL, about 8 pg/mL to about 30 pg/mL, about 8 pg/mL to about 25pg/mL, about 8 pg/mL to about 20 pg/mL, about 8 pg/mL to about 15 pg/mL,about 8 pg/mL to about 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9pg/mL to about 100 pg/mL, about 9 pg/mL to about 90 pg/mL, about 9 pg/mLto about 80 pg/mL, about 9 pg/mL to about 70 pg/mL, about 9 pg/mL toabout 60 pg/mL, about 9 pg/mL to about 55 pg/mL, about 9 pg/mL to about50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about 40pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30 pg/mL,about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL, about9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about 10pg/mL to about 100 pg/mL, about 10 pg/mL to about 90 pg/mL, about 10pg/mL to about 80 pg/mL, about 10 pg/mL to about 70 pg/mL, about 10pg/mL to about 60 pg/mL, about 10 pg/mL to about 55 pg/mL, about 10pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 100 pg/mL, about 20 pg/mL to about 90 pg/mL, about 20pg/mL to about 80 pg/mL, about 20 pg/mL to about 70 pg/mL, about 20pg/mL to about 60 pg/mL, about 20 pg/mL to about 55 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the amount of cTnI can be at least about 0.5 pg/mL, atleast about 1.0 pg/mL, at least about 1.5 pg/mL, at least about 2.0pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, at leastabout 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0 pg/mL, atleast about 7.0 pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, at least about20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL, at leastabout 35 pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, atleast about 50 pg/mL, at least about 60 pg/mL, at least about 70 pg/mL,at least about 80 pg/mL, at least about 90 pg/mL, or at least about 100pg/mL.

In some embodiments, the amount of the early biomarker, such as UCH-L1,GFAP, or a combination thereof, can be between at least about 1 pg/mL toabout 1000 pg/mL. In some embodiments, the reference level of the earlybiomarker, such as UCH-L1, GFAP, or a combination thereof, can bebetween at least about 1 pg/mL to about 1000 pg/mL, between at leastabout 1 pg/mL to about 900 pg/mL, between at least about 1 pg/mL toabout 800 pg/mL, between at least about 1 pg/mL to about 700 pg/mL,between at least about 1 pg/mL to about 600 pg/mL, between at leastabout 1 pg/mL to about 550 pg/mL, between at least about 1 pg/mL toabout 500 pg/mL, between at least about 1 pg/mL to about 450 pg/mL,between at least about 1 pg/mL to about 400 pg/mL, between at leastabout 1 pg/mL to about 300 pg/mL, between at least about 1 pg/mL toabout 300 pg/mL, between at least about 1 pg/mL to about 200 pg/mL,between at least about 1 pg/mL to about 100 pg/mL, between at leastabout 1 pg/mL to about 50 pg/mL, between at least about 1 pg/mL to about20 pg/mL, between at least about 1 pg/mL to about 15 pg/mL, between atleast about 1 pg/mL to about 10 pg/mL, between at least about 5 pg/mL toabout 1000 pg/mL, between at least about 5 pg/mL to about 900 pg/mL,between at least about 5 pg/mL to about 800 pg/mL, between at leastabout 5 pg/mL to about 700 pg/mL, between at least about 5 pg/mL toabout 600 pg/mL, between at least about 5 pg/mL to about 550 pg/mL,between at least about 5 pg/mL to about 500 pg/mL, between at leastabout 5 pg/mL to about 450 pg/mL, between at least about 5 pg/mL toabout 400 pg/mL, between at least about 5 pg/mL to about 300 pg/mL,between at least about 5 pg/mL to about 300 pg/mL, between at leastabout 5 pg/mL to about 200 pg/mL, between at least about 5 pg/mL toabout 100 pg/mL, between at least about 5 pg/mL to about 50 pg/mL,between at least about 5 pg/mL to about 20 pg/mL, between at least about5 pg/mL to about 15 pg/mL, between at least about 5 pg/mL to about 10pg/mL, between at least about 10 pg/mL to about 1000 pg/mL, between atleast about 10 pg/mL to about 900 pg/mL, between at least about 10 pg/mLto about 800 pg/mL, between at least about 10 pg/mL to about 700 pg/mL,between at least about 10 pg/mL to about 600 pg/mL, between at leastabout 10 pg/mL to about 550 pg/mL, between at least about 10 pg/mL toabout 500 pg/mL, between at least about 10 pg/mL to about 450 pg/mL,between at least about 10 pg/mL to about 400 pg/mL, between at leastabout 10 pg/mL to about 300 pg/mL, between at least about 10 pg/mL toabout 200 pg/mL, between at least about 10 pg/mL to about 100 pg/mL,between at least about 10 pg/mL to about 50 pg/mL, between at leastabout 10 pg/mL to about 20 pg/mL, between at least about 10 pg/mL toabout 15 pg/mL, between at least about 50 pg/mL to about 1000 pg/mL,between at least about 50 pg/mL to about 900 pg/mL, between at leastabout 50 pg/mL to about 800 pg/mL, between at least about 50 pg/mL toabout 700 pg/mL, between at least about 50 pg/mL to about 600 pg/mL,between at least about 50 pg/mL to about 550 pg/mL, between at leastabout 50 pg/mL to about 500 pg/mL, between at least about 50 pg/mL toabout 450 pg/mL, between at least about 50 pg/mL to about 400 pg/mL,between at least about 50 pg/mL to about 300 pg/mL, between at leastabout 50 pg/mL to about 200 pg/mL, between at least about 50 pg/mL toabout 100 pg/mL, between at least about 100 pg/mL to about 1000 pg/mL,between at least about 100 pg/mL to about 900 pg/mL, between at leastabout 100 pg/mL to about 800 pg/mL, between at least about 100 pg/mL toabout 700 pg/mL, between at least about 100 pg/mL to about 600 pg/mL,between at least about 100 pg/mL to about 550 pg/mL, between at leastabout 100 pg/mL to about 500 pg/mL, between at least about 100 pg/mL toabout 450 pg/mL, between at least about 100 pg/mL to about 400 pg/mL,between at least about 100 pg/mL to about 300 pg/mL, between at leastabout 100 pg/mL to about 200 pg/mL, between at least about 200 pg/mL toabout 1000 pg/mL, between at least about 200 pg/mL to about 900 pg/mL,between at least about 200 pg/mL to about 800 pg/mL, between at leastabout 200 pg/mL to about 700 pg/mL, between at least about 200 pg/mL toabout 600 pg/mL, between at least about 200 pg/mL to about 550 pg/mL,between at least about 200 pg/mL to about 500 pg/mL, between at leastabout 200 pg/mL to about 450 pg/mL, between at least about 200 pg/mL toabout 400 pg/mL, between at least about 200 pg/mL to about 300 pg/mL,between at least about 300 pg/mL to about 1000 pg/mL, between at leastabout 300 pg/mL to about 900 pg/mL, between at least about 300 pg/mL toabout 800 pg/mL, between at least about 300 pg/mL to about 700 pg/mL,between at least about 300 pg/mL to about 600 pg/mL, between at leastabout 300 pg/mL to about 550 pg/mL, between at least about 300 pg/mL toabout 500 pg/mL, between at least about 300 pg/mL to about 450 pg/mL,between at least about 300 pg/mL to about 400 pg/mL, between at leastabout 400 pg/mL to about 1000 pg/mL, between at least about 400 pg/mL toabout 900 pg/mL, between at least about 400 pg/mL to about 800 pg/mL,between at least about 400 pg/mL to about 700 pg/mL, between at leastabout 400 pg/mL to about 600 pg/mL, between at least about 400 pg/mL toabout 550 pg/mL, between at least about 400 pg/mL to about 500 pg/mL,between at least about 400 pg/mL to about 450 pg/mL, between at leastabout 500 pg/mL to about 1000 pg/mL, between at least about 500 pg/mL toabout 900 pg/mL, between at least about 500 pg/mL to about 800 pg/mL,between at least about 500 pg/mL to about 700 pg/mL, between at leastabout 500 pg/mL to about 600 pg/mL, between at least about 500 pg/mL toabout 550 pg/mL, between at least about 600 pg/mL to about 1000 pg/mL,between at least about 600 pg/mL to about 900 pg/mL, between at leastabout 600 pg/mL to about 800 pg/mL, or between at least about 600 pg/mLto about 700 pg/mL. In some embodiments, the amount of the earlybiomarker, such as UCH-L1, GFAP, or a combination thereof, can be atleast about 0.5 pg/mL, at least about 1.0 pg/mL, at least about 1.5pg/mL, at least about 2.0 pg/mL, at least about 2.5 pg/mL, at leastabout 3.0 pg/mL, at least about 4.0 pg/mL, at least about 5.0 pg/mL, atleast about 6.0 pg/mL, at least about 7.0 pg/mL, at least about 8.0,pg/mL, at least about 9.0 pg/mL, at least about 10 pg/mL, at least about15 pg/mL, at least about 20 pg/mL, at least about 25 pg/mL, at leastabout 30 pg/mL, at least about 35 pg/mL, at least about 40 pg/mL, atleast about 45 pg/mL, at least about 50 pg/mL, at least about 100 pg/mL,at least about 150 pg/mL, at least about 200 pg/mL, at least about 250pg/mL, at least about 300 pg/mL, at least about 350 pg/mL, at leastabout 400 pg/mL, at least about 450 pg/mL, at least about 500 pg/mL, atleast about 550 pg/mL, at least about 600 pg/mL, at least about 650pg/mL, at least about 700 pg/mL, at least about 750 pg/mL, at leastabout 800 pg/mL, at least about 850 pg/mL, at least about 900 pg/mL, atleast about 950 pg/mL, or at least about 1000 pg/mL.

In some embodiments of the above method, the reference level for cTnI,UCH-L1 and/or GFAP are as shown in the below Tables A, B and C:

TABLE A cTnI UCH-L1  About 5 pg/mL About 400 pg/mL  About 5 pg/mL About500 pg/mL  About 5 pg/mL About 550 pg/mL About 10 pg/mL About 400 pg/mLAbout 10 pg/mL About 500 pg/mL About 10 pg/mL About 550 pg/mL About 15pg/mL About 400 pg/mL About 15 pg/mL About 500 pg/mL About 15 pg/mLAbout 550 pg/mL About 20 pg/mL About 400 pg/mL About 20 pg/mL About 500pg/mL About 20 pg/mL About 550 pg/mL About 35 pg/mL About 400 pg/mLAbout 35 pg/mL About 500 pg/mL About 35 pg/mL About 550 pg/mL About 50pg/mL About 400 pg/mL About 50 pg/mL About 500 pg/mL About 50 pg/mLAbout 550 pg/mL

TABLE B cTnI GFAP  About 5 pg/mL  About 70 pg/mL  About 5 pg/mL About100 pg/mL  About 5 pg/mL About 150 pg/mL About 10 pg/mL  About 70 pg/mLAbout 10 pg/mL About 100 pg/mL About 10 pg/mL About 150 pg/mL About 15pg/mL  About 70 pg/mL About 15 pg/mL About 100 pg/mL About 15 pg/mLAbout 150 pg/mL About 20 pg/mL  About 70 pg/mL About 20 pg/mL About 100pg/mL About 20 pg/mL About 150 pg/mL About 35 pg/mL  About 70 pg/mLAbout 35 pg/mL About 100 pg/mL About 35 pg/mL About 150 pg/mL About 50pg/mL  About 70 pg/mL About 50 pg/mL About 100 pg/mL About 50 pg/mLAbout 150 pg/mL

TABLE C cTnI UCH-L1 GFAP  About 5 pg/mL About 400 pg/mL About 70 pg/mL About 5 pg/mL About 400 pg/mL About 100 pg/mL  About 5 pg/mL About 400pg/mL About 150 pg/mL  About 5 pg/mL About 500 pg/mL About 70 pg/mL About 5 pg/mL About 500 pg/mL About 100 pg/mL  About 5 pg/mL About 500pg/mL About 150 pg/mL  About 5 pg/mL About 550 pg/mL About 70 pg/mL About 5 pg/mL About 550 pg/mL About 100 pg/mL  About 5 pg/mL About 500pg/mL About 150 pg/mL About 10 pg/mL About 400 pg/mL About 70 pg/mLAbout 10 pg/mL About 400 pg/mL About 100 pg/mL About 10 pg/mL About 400pg/mL About 150 pg/mL About 10 pg/mL About 500 pg/mL About 70 pg/mLAbout 10 pg/mL About 500 pg/mL About 100 pg/mL About 10 pg/mL About 500pg/mL About 150 pg/mL About 10 pg/mL About 550 pg/mL About 70 pg/mLAbout 10 pg/mL About 550 pg/mL About 100 pg/mL About 10 pg/mL About 500pg/mL About 150 pg/mL About 15 pg/mL About 400 pg/mL About 70 pg/mLAbout 15 pg/mL About 400 pg/mL About 100 pg/mL About 15 pg/mL About 400pg/mL About 150 pg/mL About 15 pg/mL About 500 pg/mL About 70 pg/mLAbout 15 pg/mL About 500 pg/mL About 100 pg/mL About 15 pg/mL About 500pg/mL About 150 pg/mL About 15 pg/mL About 550 pg/mL About 70 pg/mLAbout 15 pg/mL About 550 pg/mL About 100 pg/mL About 15 pg/mL About 500pg/mL About 150 pg/mL About 20 pg/mL About 400 pg/mL About 70 pg/mLAbout 20 pg/mL About 400 pg/mL About 100 pg/mL About 20 pg/mL About 400pg/mL About 150 pg/mL About 20 pg/mL About 500 pg/mL About 70 pg/mLAbout 20 pg/mL About 500 pg/mL About 100 pg/mL About 20 pg/mL About 500pg/mL About 150 pg/mL About 20 pg/mL About 550 pg/mL About 70 pg/mLAbout 20 pg/mL About 550 pg/mL About 100 pg/mL About 20 pg/mL About 500pg/mL About 150 pg/mL About 35 pg/mL About 400 pg/mL About 70 pg/mLAbout 35 pg/mL About 400 pg/mL About 100 pg/mL About 35 pg/mL About 400pg/mL About 150 pg/mL About 35 pg/mL About 500 pg/mL About 70 pg/mLAbout 35 pg/mL About 500 pg/mL About 100 pg/mL About 35 pg/mL About 500pg/mL About 150 pg/mL About 35 pg/mL About 550 pg/mL About 70 pg/mLAbout 35 pg/mL About 550 pg/mL About 100 pg/mL About 35 pg/mL About 500pg/mL About 150 pg/mL About 50 pg/mL About 400 pg/mL About 70 pg/mLAbout 50 pg/mL About 400 pg/mL About 100 pg/mL About 50 pg/mL About 400pg/mL About 150 pg/mL About 50 pg/mL About 500 pg/mL About 70 pg/mLAbout 50 pg/mL About 500 pg/mL About 100 pg/mL About 50 pg/mL About 500pg/mL About 150 pg/mL About 50 pg/mL About 550 pg/mL About 70 pg/mLAbout 50 pg/mL About 550 pg/mL About 100 pg/mL About 50 pg/mL About 500pg/mL About 150 pg/mL

In some embodiments of the above method, the reference level for cTnI,UCH-L1 and/or GFAP are as shown in the below Tables D, E and F:

TABLE D CTnI UCH-L1  About 5 pg/mL About 400 pg/mL  About 5 pg/mL About500 pg/mL  About 5 pg/mL About 550 pg/mL About 10 pg/mL About 400 pg/mLAbout 10 pg/mL About 500 pg/mL About 10 pg/mL About 550 pg/mL About 15pg/mL About 400 pg/mL About 15 pg/mL About 500 pg/mL About 15 pg/mLAbout 550 pg/mL About 20 pg/mL About 400 pg/mL About 20 pg/mL About 500pg/mL About 20 pg/mL About 550 pg/mL About 35 pg/mL About 400 pg/mLAbout 35 pg/mL About 500 pg/mL About 35 pg/mL About 550 pg/mL About 50pg/mL About 400 pg/mL About 50 pg/mL About 500 pg/mL About 50 pg/mLAbout 550 pg/mL

TABLE E cTnI GFAP  About 5 pg/mL About 50 pg/mL  About 5 pg/mL About 100pg/mL  About 5 pg/mL About 150 pg/mL About 10 pg/mL About 50 pg/mL About10 pg/mL About 100 pg/mL About 10 pg/mL About 150 pg/mL About 15 pg/mLAbout 50 pg/mL About 15 pg/mL About 100 pg/mL About 15 pg/mL About 150pg/mL About 20 pg/mL About 50 pg/mL About 20 pg/mL About 100 pg/mL About20 pg/mL About 150 pg/mL About 35 pg/mL About 50 pg/mL About 35 pg/mLAbout 100 pg/mL About 35 pg/mL About 150 pg/mL About 50 pg/mL About 50pg/mL About 50 pg/mL About 100 pg/mL About 50 pg/mL About 150 pg/mL

TABLE F cTnI UCH-L1 GFAP  About 5 pg/mL About 400 pg/mL About 50 pg/mL About 5 pg/mL About 400 pg/mL About 100 pg/mL  About 5 pg/mL About 400pg/mL About 150 pg/mL  About 5 pg/mL About 500 pg/mL About 50 pg/mL About 5 pg/mL About 500 pg/mL About 100 pg/mL  About 5 pg/mL About 500pg/mL About 150 pg/mL  About 5 pg/mL About 550 pg/mL About 50 pg/mL About 5 pg/mL About 550 pg/mL About 100 pg/mL  About 5 pg/mL About 500pg/mL About 150 pg/mL About 10 pg/mL About 400 pg/mL About 50 pg/mLAbout 10 pg/mL About 400 pg/mL About 100 pg/mL About 10 pg/mL About 400pg/mL About 150 pg/mL About 10 pg/mL About 500 pg/mL About 50 pg/mLAbout 10 pg/mL About 500 pg/mL About 100 pg/mL About 10 pg/mL About 500pg/mL About 150 pg/mL About 10 pg/mL About 550 pg/mL About 50 pg/mLAbout 10 pg/mL About 550 pg/mL About 100 pg/mL About 10 pg/mL About 500pg/mL About 150 pg/mL About 15 pg/mL About 400 pg/mL About 50 pg/mLAbout 15 pg/mL About 400 pg/mL About 100 pg/mL About 15 pg/mL About 400pg/mL About 150 pg/mL About 15 pg/mL About 500 pg/mL About 50 pg/mLAbout 15 pg/mL About 500 pg/mL About 100 pg/mL About 15 pg/mL About 500pg/mL About 150 pg/mL About 15 pg/mL About 550 pg/mL About 50 pg/mLAbout 15 pg/mL About 550 pg/mL About 100 pg/mL About 15 pg/mL About 500pg/mL About 150 pg/mL About 20 pg/mL About 400 pg/mL About 50 pg/mLAbout 20 pg/mL About 400 pg/mL About 100 pg/mL About 20 pg/mL About 400pg/mL About 150 pg/mL About 20 pg/mL About 500 pg/mL About 50 pg/mLAbout 20 pg/mL About 500 pg/mL About 100 pg/mL About 20 pg/mL About 500pg/mL About 150 pg/mL About 20 pg/mL About 550 pg/mL About 50 pg/mLAbout 20 pg/mL About 550 pg/mL About 100 pg/mL About 20 pg/mL About 500pg/mL About 150 pg/mL About 35 pg/mL About 400 pg/mL About 50 pg/mLAbout 35 pg/mL About 400 pg/mL About 100 pg/mL About 35 pg/mL About 400pg/mL About 150 pg/mL About 35 pg/mL About 500 pg/mL About 50 pg/mLAbout 35 pg/mL About 500 pg/mL About 100 pg/mL About 35 pg/mL About 500pg/mL About 150 pg/mL About 35 pg/mL About 550 pg/mL About 50 pg/mLAbout 35 pg/mL About 550 pg/mL About 100 pg/mL About 35 pg/mL About 500pg/mL About 150 pg/mL About 50 pg/mL About 400 pg/mL About 50 pg/mLAbout 50 pg/mL About 400 pg/mL About 100 pg/mL About 50 pg/mL About 400pg/mL About 150 pg/mL About 50 pg/mL About 500 pg/mL About 50 pg/mLAbout 50 pg/mL About 500 pg/mL About 100 pg/mL About 50 pg/mL About 500pg/mL About 150 pg/mL About 50 pg/mL About 550 pg/mL About 50 pg/mLAbout 50 pg/mL About 550 pg/mL About 100 pg/mL About 50 pg/mL About 500pg/mL About 150 pg/mL

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject who was determined to have a TBI with a traumatic brain injurytreatment, as described below. In some embodiments, the method furtherincludes monitoring, as described below, the human subject who wasdetermined to have a TBI. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 8-12.

4. METHODS OF AIDING IN THE DIAGNOSIS AND EVALUATION OF WHETHER A HUMANSUBJECT MAY HAVE OR HAS SUSTAINED AN (OR HAS AN ACTUAL OR SUSPECTED)INJURY TO THE HEAD BASED ON CHANGES IN CARDIAC TROPONIN I (CTNI) LEVELSAND EARLY BIOMARKER LEVELS

The present disclosure relates, among other methods, to a method ofaiding in the diagnosis and evaluation of whether a human subject hassustained or may have sustained an (or has an actual or suspected)injury to the head. The method can aid in determining the extent oftraumatic brain injury in a human subject with an actual or suspectedinjury to the head, e.g., determining whether the subject has mildtraumatic brain injury or a moderate, severe, or moderate to severetraumatic brain injury. As used here, “determining whether the subjecthas mild traumatic brain injury or a moderate, severe, or moderate tosevere traumatic brain injury” refers to the fact that theaforementioned method can be used, e.g., with other information (e.g.,clinical assessment data), to determine that the subject is more likelythan not to have mild traumatic brain injury or moderate to severetraumatic brain injury. The method can include performing an assay on atleast two samples obtained from the subject, the first sample taken fromthe subject within about 24 hours, such as within about 2 hours, afteran actual or suspected injury to the head and the second sample takenfrom the subject from about 3 to about 6 hours after the first sample istaken; detecting in the at least two samples cardiac troponin I (cTnI)and a level of an early biomarker; and determining whether the subjecthas sustained a mild or a moderate, severe, or moderate to severetraumatic brain injury (TBI). The subject is determined as having (1) amoderate, severe, or a moderate to severe traumatic brain injury whenthe level of cTnI decreases or increases by at least an absolute amountfrom the first sample to the second sample and the level of the earlybiomarker decreases or increases by at least an absolute amount from thefirst sample to the second sample or (2) a mild traumatic brain injurywhen there is no decrease or increase by at least an absolute amount inthe level of cTnI from the first sample to the second sample and/orthere is no decrease or increase by at least an absolute amount in thelevel of the early biomarker from the first sample to the second sample.The samples can be biological samples. The early biomarker includesubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillaryacidic protein (GFAP), or a combination thereof.

In an alternative, the method can include performing an assay on atleast two samples obtained from the subject, the first sample taken fromthe subject within about 24 hours, such as within about 2 hours, afteran actual or suspected injury to the head and the second sample takenfrom the subject from about 3 to about 6 hours after the first sample istaken; detecting in the at least two samples cTnI and the earlybiomarker; and determining whether the subject has sustained a mild or amoderate, severe, or moderate to severe traumatic brain injury (TBI),wherein the subject is determined as having (1) a moderate, severe, or amoderate to severe traumatic brain injury when the level of cTnIdecreases or increases by at least a first absolute amount from thefirst sample to the second sample and the level of the early biomarkerdecreases or increases by at least a first absolute amount from thefirst sample to the second sample or (2) a mild traumatic brain injurywhen there is no decrease or increase by at least a second absoluteamount in the level of cTnI from the first sample to the second sampleand/or there is no decrease or increase by at least a second absoluteamount in the level of and the early biomarker from the first sample tothe second sample. The samples can be biological samples.

In some embodiments, the method can include contacting the samples withan antibody for cTnI, to allow formation of a complex of the antibodyand cTnI. The method also includes detecting the resulting antibody-cTnIcomplex to determine the levels of cTnI for each of the first sample andsecond sample. The onset of the presence of cTnI appears within about 0to about 2 hours after the onset of the suspected injury. In someembodiments, the onset of the presence of cTnI appears within about 0minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about2 hours after injury to the head.

In some embodiments, the first sample is obtained at a first time pointwithin about 24 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 24 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample may beobtained or taken from the subject within about 0 minutes, within about1 minute, within about 2 minutes, within about 3 minutes, within about 4minutes, within about 5 minutes, within about 6 minutes, within about 7minutes, within about 8 minutes, within about 9 minutes, within about 10minutes, within about 11 minutes, within about 12 minutes, within about13 minutes, within about 14 minutes, within about 15 minutes, withinabout 20 minutes, within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours of a suspect injury to the head.

In some embodiments, the first sample is obtained at a first time pointwithin about 2 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 2 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample is takenabout 0 to about 2 hours after the injury or suspected injury to thehead. For example, the first sample can be taken between about 0 toabout 2 hours, about 0 hours to about 90 minutes, about 0 hours to about60 minutes, about 0 hours to about 45 minutes, about 0 hours to about 30minutes, about 0 hours to about 20 minutes, about 0 hours to about 15minutes, about 0 hours to about 10 minutes, about 0 hours to about 5minutes, about 5 minutes to about 90 minutes, about 5 minutes to about60 minutes, about 5 minutes to about 45 minutes, about 5 minutes toabout 30 minutes, about 5 minutes to about 20 minutes, about 5 minutesto about 15 minutes, about 5 minutes to about 10 minutes, about 10minutes to about 90 minutes, about 10 minutes to about 60 minutes, about10 minutes to about 45 minutes, about 10 minutes to about 30 minutes,about 10 minutes to about 20 minutes, about 10 minutes to about 15minutes, about 15 minutes to about 90 minutes, about 15 minutes to about60 minutes, about 15 minutes to about 45 minutes, about 15 minutes toabout 30 minutes, about 15 minutes to about 20 minutes, about 20 minutesto about 90 minutes, about 20 minutes to about 60 minutes, about 20minutes to about 45 minutes, or about 20 minutes to about 30 minutesafter the suspected injury. For example, the first sample can be takenfrom the human subject within about 0 minutes, about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60minutes, about 90 minutes, or about 2 hours of injury or suspectedinjury to the head.

In some embodiments, the second sample is taken about 1 hour to about 10hours after the first time point, such as about 3 hours to about 6 hoursafter the first time point. In some embodiments, the second sample istaken about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, orabout 10 hours after the first sample.

In some embodiments, the subject may have received a Glasgow Coma Scalescore before or after the level of cTnI is determined at one or moretime points. In certain embodiments, the subject may be suspected ofhaving a mild traumatic brain injury based on the Glasgow Coma Scalescore. In certain embodiments, the subject may be suspected of having amild traumatic brain injury based on an abnormal head CT. In someembodiments, the subject has received a CT scan before or after theassay is performed. In some embodiments, the subject has a normal headCT.

In some embodiments, the reference level of cTnI is correlated withsubjects having a moderate, severe, or moderate to severe traumaticbrain injury. In some embodiments, the reference level of cTnI iscorrelated with a Glasgow Coma Scale score of 3-12 (moderate to severeTBI). In some embodiments, the reference level of cTnI is correlatedwith a Glasgow Coma Scale score of 3-8 (a severe TBI). In someembodiments, the reference level of cTnI is correlated with a GlasgowComa Scale score of 9-13 (a moderate TBI). In some embodiments, thesubject is suspected as having mild traumatic brain injury based on theGlasgow Coma Scale score. In some embodiments, the reference level ofcTnI is correlated with subjects having mild traumatic brain injury. Insome embodiments, the reference level of cTnI is correlated with aGlasgow Coma Scale score of 13-15 (a mild TBI).

In some embodiments, the absolute amount can be determined by an assayhaving a sensitivity of between at least about 65% to about 100% and aspecificity of between at least about 65% to about 100%. For example,the absolute amount can be determined by an assay having a sensitivityof between at least about 80% to 100% and a specificity of between atleast about 65% to 100%. In some embodiments, the sensitivity is atleast about 65.0%, the sensitivity is at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%. In someembodiments, the specificity is at least about 65.0%, at least about70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%,at least about 90.0%, at least about 91.0%, at least about 92.0%, atleast about 93. %, at least about 94.0%, at least about 95.0%, at leastabout 96.0%, at least about 97.0%, at least about 98.0%, at least about99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%,at least about 99.4%, at least about 99.5%, at least about 99.6%, atleast about 99.7%, at least about 99.8%, at least about 99.9%, or atleast about 100.0%. For example, the sensitivity is at least about 100%and the specificity is at least about 75%, the sensitivity is at leastabout 99% and the specificity is at least about 99%, or the sensitivityis at least about 87% and the specificity is at least about 95%.

In some embodiments, the absolute amount of cTnI in the sample is fromabout 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about 45 pg/mL, about1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35 pg/mL, about 1pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL, about 1 pg/mLto about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about 1 pg/mL toabout 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mL to about 8pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about 6 pg/mL,about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL, about 1pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1 pg/mL toabout 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5 pg/mL toabout 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5 pg/mL toabout 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5 pg/mL toabout 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5 pg/mL toabout 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5 pg/mL toabout 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5 pg/mL toabout 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5 pg/mL toabout 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5 pg/mL toabout 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mL to about50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL to about 40pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about 30 pg/mL,about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20 pg/mL, about2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL, about 2pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2 pg/mL toabout 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL to about 5pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3 pg/mL,about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL, about3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about 3pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3 pg/mLto about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mL toabout 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL to about 8pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6 pg/mL,about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL, about 4pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about 4 pg/mLto about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4 pg/mL toabout 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mL to about20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL to about 10pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8 pg/mL,about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL, about 4pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5 pg/mLto about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mL toabout 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL to about25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about 15pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9 pg/mL,about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL, about 5pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6 pg/mLto about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mL toabout 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL to about25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about 15pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9 pg/mL,about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL, about 7pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about 7 pg/mLto about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7 pg/mL toabout 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mL to about20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL to about 10pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8 pg/mL,about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL, about8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about 8pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8 pg/mLto about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mL toabout 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL to about50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about 40pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30 pg/mL,about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL, about9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about 10pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the absolute amount of cTnI can be at least about 0.5pg/mL, at least about 1.0 pg/mL, at least about 1.5 pg/mL, at leastabout 2.0 pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, atleast about 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0pg/mL, at least about 7.0 pg/mL, at least about 8.0, pg/mL, at leastabout 9.0 pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, atleast about 20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL,at least about 35 pg/mL, at least about 40 pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In some embodiments, the absolute amount of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, can be between at least about 5pg/mL to about 1000 pg/mL. In some embodiments, the absolute amount canbe between at least about 5 pg/mL to about 1000 pg/mL, between at leastabout 5 pg/mL to about 750 pg/mL, between at least about 5 pg/mL toabout 500 pg/mL, between at least about 5 pg/mL to about 400 pg/mL,between at least about 5 pg/mL to about 300 pg/mL, between at leastabout 5 pg/mL to about 200 pg/mL, between at least about 5 pg/mL toabout 100 pg/mL, between at least about 5 pg/mL to about 50 pg/mL,between at least about 10 pg/mL to about 1000 pg/mL, between at leastabout 10 pg/mL to about 750 pg/mL, between at least about 10 pg/mL toabout 500 pg/mL, between at least about 10 pg/mL to about 400 pg/mL,between at least about 10 pg/mL to about 300 pg/mL, between at leastabout 10 pg/mL to about 200 pg/mL, between at least about 10 pg/mL toabout 100 pg/mL, between at least about 10 pg/mL to about 50 pg/mL,between at least about 20 pg/mL to about 1000 pg/mL, between at leastabout 20 pg/mL to about 750 pg/mL, between at least about 20 pg/mL toabout 500 pg/mL, between at least about 20 pg/mL to about 400 pg/mL,between at least about 20 pg/mL to about 300 pg/mL, between at leastabout 20 pg/mL to about 200 pg/mL, between at least about 20 pg/mL toabout 100 pg/mL, between at least about 20 pg/mL to about 50 pg/mL,between at least about 25 pg/mL to about 1000 pg/mL, between at leastabout 25 pg/mL to about 750 pg/mL, between at least about 25 pg/mL toabout 500 pg/mL, between at least about 25 pg/mL to about 400 pg/mL,between at least about 25 pg/mL to about 300 pg/mL, between at leastabout 25 pg/mL to about 200 pg/mL, between at least about 25 pg/mL toabout 100 pg/mL, between at least about 25 pg/mL to about 50 pg/mL,between at least about 50 pg/mL to about 1000 pg/mL, between at leastabout 50 pg/mL to about 750 pg/mL, between at least about 50 pg/mL toabout 500 pg/mL, between at least about 50 pg/mL to about 400 pg/mL,between at least about 50 pg/mL to about 300 pg/mL, between at leastabout 50 pg/mL to about 200 pg/mL, between at least about 50 pg/mL toabout 100 pg/mL, between at least about 100 pg/mL to about 1000 pg/mL,between at least about 100 pg/mL to about 750 pg/mL, between at leastabout 100 pg/mL to about 500 pg/mL, between at least about 100 pg/mL toabout 400 pg/mL, between at least about 100 pg/mL to about 300 pg/mL,between at least about 100 pg/mL to about 200 pg/mL, between at leastabout 200 pg/mL to about 1000 pg/mL, between at least about 200 pg/mL toabout 750 pg/mL, between at least about 200 pg/mL to about 500 pg/mL,between at least about 200 pg/mL to about 400 pg/mL, between at leastabout 200 pg/mL to about 300 pg/mL, between at least about 300 pg/mL toabout 1000 pg/mL, between at least about 300 pg/mL to about 750 pg/mL,between at least about 300 pg/mL to about 500 pg/mL, between at leastabout 300 pg/mL to about 400 pg/mL, between at least about 400 pg/mL toabout 1000 pg/mL, between at least about 400 pg/mL to about 750 pg/mL,or between at least about 400 pg/mL to about 500 pg/mL. In someembodiments, the absolute amount can be at least about 5 pg/mL, at leastabout 6 pg/mL, at least about 7 pg/mL, at least about 8 pg/mL, at leastabout 9 pg/mL, at least about 10 pg/mL, at least about 11 pg/mL, atleast about 12 pg/mL, at least about 13 pg/mL, at least about 14 pg/mL,at least about 15 pg/mL, at least about 16 pg/mL, at least about 17pg/mL, at least about 18 pg/mL, at least about 19 pg/mL, at least about20 pg/mL, at least about 21 pg/mL, at least about 22 pg/mL, at leastabout 23 pg/mL, at least about 24 pg/mL, at least about 25 pg/mL, atleast about 26 pg/mL, at least about 27 pg/mL, at least about 28 pg/mL,at least about 29 pg/mL, at least about 30 pg/mL, at least about 35pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, at least about50 pg/mL, at least about 55 pg/mL, at least about 60 pg/mL, at leastabout 65 pg/mL, at least about 70 pg/mL, at least about 75 pg/mL, atleast about 80 pg/mL, at least about 85 pg/mL, at least about 90 pg/mL,at least about 95 pg/mL, at least about 100 pg/mL, at least about 110pg/mL, at least about 120 pg/mL, at least about 129 pg/mL, at leastabout 130 pg/mL, at least about 140 pg/mL, at least about 150 pg/mL, atleast about 200 pg/mL, at least about 250 pg/mL, at least about 300pg/mL, at least about 350 pg/mL, at least about 400 pg/mL, at leastabout 450 pg/mL, at least about 500 pg/mL, at least about 550 pg/mL, atleast about 600 pg/mL, at least about 650 pg/mL, at least about 700pg/mL, at least about 750 pg/mL, at least about 800 pg/mL, at leastabout 900 pg/mL, or at least about 1000 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject assessed as having a moderate, severe, or moderate to severetraumatic brain injury with a traumatic brain injury treatment, asdescribed below. In some embodiments, the method further includesmonitoring the human subject assessed as having mild traumatic braininjury, as described below. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical, and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 8-12.

5. METHOD OF AIDING IN THE DETERMINATION OF WHETHER TO PERFORM A CT SCANON A HUMAN SUBJECT WHO MAY HAVE OR HAS SUSTAINED AN (OR HAS AN ACTUAL ORSUSPECTED) INJURY TO THE HEAD BASED ON CHANGES IN CARDIAC TROPONIN I(CTNI) LEVELS AND EARLY BIOMARKER LEVELS

The present disclosure relates, among other methods, to a method ofaiding in determining whether to perform a computerized tomography (CT)scan on a human subject who has sustained or may have sustained an (orhas an actual or suspected) injury to the head. As used here,“determination of whether to perform a CT scan on a human subject”refers to the fact that the aforementioned method can be used, e.g.,with other information (e.g., clinical assessment data), to determinethat the subject is more likely than not to have a positive head CTscan. Specifically, such a method can comprise the steps of: performingan assay on at least two samples obtained from the subject, the firstsample taken from the subject within about 24 hours, such as withinabout 2 hours, of the suspected injury and the second sample taken fromthe subject from about 3 to about 6 hours after the first sample istaken; detecting in the at least two samples cardiac troponin I (cTnI)and a level of an early biomarker; and performing a CT scan on thesubject when the level of cTnI decreases or increases by at least anabsolute amount from the first sample to the second sample and the levelof the early biomarker decreases or increases by at least an absoluteamount from the first sample to the second sample and not performing aCT scan on the subject when there is no decrease or increase by at leastan absolute amount in the level of cTnI from the first sample to thesecond sample and/or there is no decrease or increase by at least anabsolute amount in the level of the early biomarker from the firstsample to the second sample. The samples can be biological samples. Theearly biomarker includes ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof.

In some embodiments, the method can include contacting the samples withan antibody for cTnI, to allow formation of a complex of the antibodyand cTnI and with an antibody for the early biomarker, to allowformation of a complex of the antibody and the early biomarker. Themethod also includes detecting the resulting antibody-cTnI complex todetermine the levels of cTnI for each of the first sample and secondsample and detecting the resulting antibody-early biomarker complex todetermine the levels of the early biomarker for each of the first sampleand second sample. The onset of the presence of cTnI and/or the earlybiomarker appears within about 0 to about 24 hours, such as within about2 hours, after the onset of the suspected injury. In some embodiments,the onset of the presence of cTnI and/or the early biomarker appearswithin about 0 minutes, about 1 minute, about 2 minutes, about 3minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90minutes, or about 2 hours after injury to the head.

In some embodiments, the first sample is obtained at a first time pointwithin about 24 hours of the suspected injury and the second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point. In some embodiments, thefirst sample is taken within about 24 hours after the suspected injuryand the second sample is taken within about 3 hours to about 6 hoursafter the first sample. In some embodiments, the first sample may beobtained or taken from the subject within about 0 minutes, within about1 minute, within about 2 minutes, within about 3 minutes, within about 4minutes, within about 5 minutes, within about 6 minutes, within about 7minutes, within about 8 minutes, within about 9 minutes, within about 10minutes, within about 11 minutes, within about 12 minutes, within about13 minutes, within about 14 minutes, within about 15 minutes, withinabout 20 minutes, within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours or within about 24 hours of a suspect injury to the head.

In some embodiments, a first sample is obtained at a first time pointwithin about 2 hours of the suspected injury and a second sample isobtained at second time point, or optionally a third time point orfourth time point, after the first time point to determine whether thesubject will have a positive or negative head CT scan. In someembodiments, the first sample is taken within about 2 hours after thesuspected injury and the second sample is taken within about 3 hours toabout 6 hours after the first sample. In some embodiments, the firsttime point is about 0 to about 2 hours after the injury or suspectedinjury to the head. For example, the first time point can be betweenabout 0 to about 2 hours, about 0 hours to about 90 minutes, about 0hours to about 60 minutes, about 0 hours to about 45 minutes, about 0hours to about 30 minutes, about 0 hours to about 20 minutes, about 0hours to about 15 minutes, about 0 hours to about 10 minutes, about 0hours to about 5 minutes, about 5 minutes to about 90 minutes, about 5minutes to about 60 minutes, about 5 minutes to about 45 minutes, about5 minutes to about 30 minutes, about 5 minutes to about 20 minutes,about 5 minutes to about 15 minutes, about 5 minutes to about 10minutes, about 10 minutes to about 90 minutes, about 10 minutes to about60 minutes, about 10 minutes to about 45 minutes, about 10 minutes toabout 30 minutes, about 10 minutes to about 20 minutes, about 10 minutesto about 15 minutes, about 15 minutes to about 90 minutes, about 15minutes to about 60 minutes, about 15 minutes to about 45 minutes, about15 minutes to about 30 minutes, about 15 minutes to about 20 minutes,about 20 minutes to about 90 minutes, about 20 minutes to about 60minutes, about 20 minutes to about 45 minutes, or about 20 minutes toabout 30 minutes after the suspected injury.

In some embodiments, the second time point, or optionally a third timepoint or fourth time point, is about 1 hour to about 10 hours after thefirst time point, such as about 3 hours to about 6 hours after the firsttime point. In some embodiments, the second time point is about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hoursafter the first time point.

In some embodiments, the absolute amount can be determined by an assayhaving a sensitivity of between at least about 65% to about 100% and aspecificity of between at least about 65% to about 100%. For example,the absolute amount can be determined by an assay having a sensitivityof between at least about 80% to 100% and a specificity of between atleast about 65% to 100%. In some embodiments, the sensitivity is atleast about 65.0%, the sensitivity is at least about 70.0%, at leastabout 75.0%, at least about 80.0%, at least about 85.0%, at least about90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%,at least about 99.2%, at least about 99.3%, at least about 99.4%, atleast about 99.5%, at least about 99.6%, at least about 99.7%, at leastabout 99.8%, at least about 99.9%, or at least about 100.0%. In someembodiments, the specificity is at least about 65.0%, at least about70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%,at least about 90.0%, at least about 91.0%, at least about 92.0%, atleast about 93. %, at least about 94.0%, at least about 95.0%, at leastabout 96.0%, at least about 97.0%, at least about 98.0%, at least about99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%,at least about 99.4%, at least about 99.5%, at least about 99.6%, atleast about 99.7%, at least about 99.8%, at least about 99.9%, or atleast about 100.0%. For example, the sensitivity is at least about 100%and the specificity is at least about 75%, the sensitivity is at leastabout 99% and the specificity is at least about 99%, or the sensitivityis at least about 87% and the specificity is at least about 95%.

In some embodiments, the absolute amount of cTnI in the sample is fromabout 1 pg/mL to about 50 pg/mL, about 1 pg/mL to about 45 pg/mL, about1 pg/mL to about 40 pg/mL, about 1 pg/mL to about 35 pg/mL, about 1pg/mL to about 30 pg/mL, about 1 pg/mL to about 25 pg/mL, about 1 pg/mLto about 20 pg/mL, about 1 pg/mL to about 15 pg/mL, about 1 pg/mL toabout 10 pg/mL, about 1 pg/mL to about 9 pg/mL, about 1 pg/mL to about 8pg/mL, about 1 pg/mL to about 7 pg/mL, about 1 pg/mL to about 6 pg/mL,about 1 pg/mL to about 5 pg/mL, about 1 pg/mL to about 4 pg/mL, about 1pg/mL to about 3 pg/mL, about 1 pg/mL to about 2 pg/mL, about 1 pg/mL toabout 1.5 pg/mL, about 1.5 pg/mL to about 50 pg/mL, about 1.5 pg/mL toabout 45 pg/mL, about 1.5 pg/mL to about 40 pg/mL, about 1.5 pg/mL toabout 35 pg/mL, about 1.5 pg/mL to about 30 pg/mL, about 1.5 pg/mL toabout 25 pg/mL, about 1.5 pg/mL to about 20 pg/mL, about 1.5 pg/mL toabout 15 pg/mL, about 1.5 pg/mL to about 10 pg/mL, about 1.5 pg/mL toabout 9 pg/mL, about 1.5 pg/mL to about 8 pg/mL, about 1.5 pg/mL toabout 7 pg/mL, about 1.5 pg/mL to about 6 pg/mL, about 1.5 pg/mL toabout 5 pg/mL, about 1.5 pg/mL to about 4 pg/mL, about 1.5 pg/mL toabout 3 pg/mL, about 1.5 pg/mL to about 2 pg/mL, about 2 pg/mL to about50 pg/mL, about 2 pg/mL to about 45 pg/mL, about 2 pg/mL to about 40pg/mL, about 2 pg/mL to about 35 pg/mL, about 2 pg/mL to about 30 pg/mL,about 2 pg/mL to about 25 pg/mL, about 2 pg/mL to about 20 pg/mL, about2 pg/mL to about 15 pg/mL, about 2 pg/mL to about 10 pg/mL, about 2pg/mL to about 9 pg/mL, about 2 pg/mL to about 8 pg/mL, about 2 pg/mL toabout 7 pg/mL, about 2 pg/mL to about 6 pg/mL, about 2 pg/mL to about 5pg/mL, about 2 pg/mL to about 4 pg/mL, about 2 pg/mL to about 3 pg/mL,about 3 pg/mL to about 50 pg/mL, about 3 pg/mL to about 45 pg/mL, about3 pg/mL to about 40 pg/mL, about 3 pg/mL to about 35 pg/mL, about 3pg/mL to about 30 pg/mL, about 3 pg/mL to about 25 pg/mL, about 3 pg/mLto about 20 pg/mL, about 3 pg/mL to about 15 pg/mL, about 3 pg/mL toabout 10 pg/mL, about 3 pg/mL to about 9 pg/mL, about 3 pg/mL to about 8pg/mL, about 3 pg/mL to about 7 pg/mL, about 3 pg/mL to about 6 pg/mL,about 3 pg/mL to about 5 pg/mL, about 3 pg/mL to about 4 pg/mL, about 4pg/mL to about 50 pg/mL, about 4 pg/mL to about 45 pg/mL, about 4 pg/mLto about 40 pg/mL, about 4 pg/mL to about 35 pg/mL, about 4 pg/mL toabout 30 pg/mL, about 4 pg/mL to about 25 pg/mL, about 4 pg/mL to about20 pg/mL, about 4 pg/mL to about 15 pg/mL, about 4 pg/mL to about 10pg/mL, about 4 pg/mL to about 9 pg/mL, about 4 pg/mL to about 8 pg/mL,about 4 pg/mL to about 7 pg/mL, about 4 pg/mL to about 6 pg/mL, about 4pg/mL to about 5 pg/mL, about 5 pg/mL to about 50 pg/mL, about 5 pg/mLto about 45 pg/mL, about 5 pg/mL to about 40 pg/mL, about 5 pg/mL toabout 35 pg/mL, about 5 pg/mL to about 30 pg/mL, about 5 pg/mL to about25 pg/mL, about 5 pg/mL to about 20 pg/mL, about 5 pg/mL to about 15pg/mL, about 5 pg/mL to about 10 pg/mL, about 5 pg/mL to about 9 pg/mL,about 5 pg/mL to about 8 pg/mL, about 5 pg/mL to about 7 pg/mL, about 5pg/mL to about 6 pg/mL, about 6 pg/mL to about 50 pg/mL, about 6 pg/mLto about 45 pg/mL, about 6 pg/mL to about 40 pg/mL, about 6 pg/mL toabout 35 pg/mL, about 6 pg/mL to about 30 pg/mL, about 6 pg/mL to about25 pg/mL, about 6 pg/mL to about 20 pg/mL, about 6 pg/mL to about 15pg/mL, about 6 pg/mL to about 10 pg/mL, about 6 pg/mL to about 9 pg/mL,about 6 pg/mL to about 8 pg/mL, about 6 pg/mL to about 7 pg/mL, about 7pg/mL to about 50 pg/mL, about 7 pg/mL to about 45 pg/mL, about 7 pg/mLto about 40 pg/mL, about 7 pg/mL to about 35 pg/mL, about 7 pg/mL toabout 30 pg/mL, about 7 pg/mL to about 25 pg/mL, about 7 pg/mL to about20 pg/mL, about 7 pg/mL to about 15 pg/mL, about 7 pg/mL to about 10pg/mL, about 7 pg/mL to about 9 pg/mL, about 7 pg/mL to about 8 pg/mL,about 8 pg/mL to about 50 pg/mL, about 8 pg/mL to about 45 pg/mL, about8 pg/mL to about 40 pg/mL, about 8 pg/mL to about 35 pg/mL, about 8pg/mL to about 30 pg/mL, about 8 pg/mL to about 25 pg/mL, about 8 pg/mLto about 20 pg/mL, about 8 pg/mL to about 15 pg/mL, about 8 pg/mL toabout 10 pg/mL, about 8 pg/mL to about 9 pg/mL, about 9 pg/mL to about50 pg/mL, about 9 pg/mL to about 45 pg/mL, about 9 pg/mL to about 40pg/mL, about 9 pg/mL to about 35 pg/mL, about 9 pg/mL to about 30 pg/mL,about 9 pg/mL to about 25 pg/mL, about 9 pg/mL to about 20 pg/mL, about9 pg/mL to about 15 pg/mL, about 9 pg/mL to about 10 pg/mL, about 10pg/mL to about 50 pg/mL, about 10 pg/mL to about 45 pg/mL, about 10pg/mL to about 40 pg/mL, about 10 pg/mL to about 35 pg/mL, about 10pg/mL to about 30 pg/mL, about 10 pg/mL to about 25 pg/mL, about 10pg/mL to about 20 pg/mL, about 10 pg/mL to about 15 pg/mL, about 20pg/mL to about 50 pg/mL, about 20 pg/mL to about 45 pg/mL, about 20pg/mL to about 40 pg/mL, about 20 pg/mL to about 35 pg/mL, about 20pg/mL to about 30 pg/mL, or about 20 pg/mL to about 25 pg/mL. In someembodiments, the absolute amount of cTnI can be at least about 0.5pg/mL, at least about 1.0 pg/mL, at least about 1.5 pg/mL, at leastabout 2.0 pg/mL, at least about 2.5 pg/mL, at least about 3.0 pg/mL, atleast about 4.0 pg/mL, at least about 5.0 pg/mL, at least about 6.0pg/mL, at least about 7.0 pg/mL, at least about 8.0, pg/mL, at leastabout 9.0 pg/mL, at least about 10 pg/mL, at least about 15 pg/mL, atleast about 20 pg/mL, at least about 25 pg/mL, at least about 30 pg/mL,at least about 35 pg/mL, at least about 40 pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In some embodiments, the absolute amount of the early biomarker, such asUCH-L1, GFAP, or a combination thereof, can be between at least about 5pg/mL to about 1000 pg/mL. In some embodiments, the absolute amount canbe between at least about 5 pg/mL to about 1000 pg/mL, between at leastabout 5 pg/mL to about 750 pg/mL, between at least about 5 pg/mL toabout 500 pg/mL, between at least about 5 pg/mL to about 400 pg/mL,between at least about 5 pg/mL to about 300 pg/mL, between at leastabout 5 pg/mL to about 200 pg/mL, between at least about 5 pg/mL toabout 100 pg/mL, between at least about 5 pg/mL to about 50 pg/mL,between at least about 10 pg/mL to about 1000 pg/mL, between at leastabout 10 pg/mL to about 750 pg/mL, between at least about 10 pg/mL toabout 500 pg/mL, between at least about 10 pg/mL to about 400 pg/mL,between at least about 10 pg/mL to about 300 pg/mL, between at leastabout 10 pg/mL to about 200 pg/mL, between at least about 10 pg/mL toabout 100 pg/mL, between at least about 10 pg/mL to about 50 pg/mL,between at least about 20 pg/mL to about 1000 pg/mL, between at leastabout 20 pg/mL to about 750 pg/mL, between at least about 20 pg/mL toabout 500 pg/mL, between at least about 20 pg/mL to about 400 pg/mL,between at least about 20 pg/mL to about 300 pg/mL, between at leastabout 20 pg/mL to about 200 pg/mL, between at least about 20 pg/mL toabout 100 pg/mL, between at least about 20 pg/mL to about 50 pg/mL,between at least about 25 pg/mL to about 1000 pg/mL, between at leastabout 25 pg/mL to about 750 pg/mL, between at least about 25 pg/mL toabout 500 pg/mL, between at least about 25 pg/mL to about 400 pg/mL,between at least about 25 pg/mL to about 300 pg/mL, between at leastabout 25 pg/mL to about 200 pg/mL, between at least about 25 pg/mL toabout 100 pg/mL, between at least about 25 pg/mL to about 50 pg/mL,between at least about 50 pg/mL to about 1000 pg/mL, between at leastabout 50 pg/mL to about 750 pg/mL, between at least about 50 pg/mL toabout 500 pg/mL, between at least about 50 pg/mL to about 400 pg/mL,between at least about 50 pg/mL to about 300 pg/mL, between at leastabout 50 pg/mL to about 200 pg/mL, between at least about 50 pg/mL toabout 100 pg/mL, between at least about 100 pg/mL to about 1000 pg/mL,between at least about 100 pg/mL to about 750 pg/mL, between at leastabout 100 pg/mL to about 500 pg/mL, between at least about 100 pg/mL toabout 400 pg/mL, between at least about 100 pg/mL to about 300 pg/mL,between at least about 100 pg/mL to about 200 pg/mL, between at leastabout 200 pg/mL to about 1000 pg/mL, between at least about 200 pg/mL toabout 750 pg/mL, between at least about 200 pg/mL to about 500 pg/mL,between at least about 200 pg/mL to about 400 pg/mL, between at leastabout 200 pg/mL to about 300 pg/mL, between at least about 300 pg/mL toabout 1000 pg/mL, between at least about 300 pg/mL to about 750 pg/mL,between at least about 300 pg/mL to about 500 pg/mL, between at leastabout 300 pg/mL to about 400 pg/mL, between at least about 400 pg/mL toabout 1000 pg/mL, between at least about 400 pg/mL to about 750 pg/mL,or between at least about 400 pg/mL to about 500 pg/mL. In someembodiments, the absolute amount can be at least about 5 pg/mL, at leastabout 6 pg/mL, at least about 7 pg/mL, at least about 8 pg/mL, at leastabout 9 pg/mL, at least about 10 pg/mL, at least about 11 pg/mL, atleast about 12 pg/mL, at least about 13 pg/mL, at least about 14 pg/mL,at least about 15 pg/mL, at least about 16 pg/mL, at least about 17pg/mL, at least about 18 pg/mL, at least about 19 pg/mL, at least about20 pg/mL, at least about 21 pg/mL, at least about 22 pg/mL, at leastabout 23 pg/mL, at least about 24 pg/mL, at least about 25 pg/mL, atleast about 26 pg/mL, at least about 27 pg/mL, at least about 28 pg/mL,at least about 29 pg/mL, at least about 30 pg/mL, at least about 35pg/mL, at least about 40 pg/mL, at least about 45 pg/mL, at least about50 pg/mL, at least about 55 pg/mL, at least about 60 pg/mL, at leastabout 65 pg/mL, at least about 70 pg/mL, at least about 75 pg/mL, atleast about 80 pg/mL, at least about 85 pg/mL, at least about 90 pg/mL,at least about 95 pg/mL, at least about 100 pg/mL, at least about 110pg/mL, at least about 120 pg/mL, at least about 129 pg/mL, at leastabout 130 pg/mL, at least about 140 pg/mL, at least about 150 pg/mL, atleast about 200 pg/mL, at least about 250 pg/mL, at least about 300pg/mL, at least about 350 pg/mL, at least about 400 pg/mL, at leastabout 450 pg/mL, at least about 500 pg/mL, at least about 550 pg/mL, atleast about 600 pg/mL, at least about 650 pg/mL, at least about 700pg/mL, at least about 750 pg/mL, at least about 800 pg/mL, at leastabout 900 pg/mL, or at least about 1000 pg/mL.

In addition to performing the above described methods, one skilled inthe art (e.g., physician) would understand and know how to performadditional testing in order to detect or assess other comorbidities(e.g., other diseases, disorders, or conditions other than TBI). Suchadditional tests or procedures include one or more of anelectrocardiogram, a complete blood cell (CBC) count, a comprehensivemetabolic panel, a lipid profile (e.g., to determine HDL, LDL,triglycerides, etc.), an angiogram, one or more tests to detect ordetermine the levels of one or more of c reactive protein (CRP), brainnatriuretic peptide, plasma ceramides, etc.

In one embodiment, in order to confirm that the changes in amounts orlevels cTnI in the methods described herein are attributable to a headinjury or a suspected injury to the head of a subject and not the resultof an acute cardiac syndrome (such as a myocardial infarction, heartfailure, etc.), a physician or other healthcare provider could conductor perform one or more additional tests or procedures to confirm theabsence of an acute cardiac syndrome. Such additional tests orprocedures include one or more of an electrocardiogram, a complete bloodcell (CBC) count, a comprehensive metabolic panel, a lipid profile(e.g., to determine HDL, LDL, triglycerides, etc.), an angiogram, one ormore tests to detect or determine the levels of one or more of creactive protein (CRP), brain natriuretic peptide, plasma ceramides,etc.

In some embodiments, the method further includes treating the humansubject who was determined to have a TBI with a traumatic brain injurytreatment, as described below. In some embodiments, the method furtherincludes monitoring, as described below, the human subject who wasdetermined to have a TBI. In some embodiments, the method furtherincludes ordering additional tests to obtain further clinicalinformation about the traumatic brain injury. In some embodiments, themethod includes treating the human subject assessed as having a mild,moderate, severe, or a moderate to severe brain injury with acardioprotective treatment to protect the heart as described below.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, theassay can be employed in clinical chemistry format such as would beknown by one skilled in the art. Such assays are described in furtherdetail herein in Sections 8-12.

6. METHODS OF TREATING A HUMAN SUBJECT HAVING TRAUMATIC BRAIN INJURYWITH CARDIOPROTECTIVE THERAPY

The present disclosure relates, among other methods, to a method fortreating a human subject having or suspected as having a traumatic braininjury. The occurrence of myocardial injury during the acute phase oftraumatic brain injury (TBI) may be a contributor to poor TBI outcome.As such, administering one or more cardioprotective therapies ortherapeutics (e.g., therapies that improve the heart) may improve TBIoutcome and can be employed in the methods described herein. Thesecardioprotective therapies can be administered alone without any othertherapeutics. Alternatively, these cardioprotective therapies can beadministered in combination with other therapeutics administered totreat the TBI, such as those disclosed in Section 7, below.

Specifically, the methods of the disclosure involving one or morecardioprotective therapies or therapeutics includes: a) performing anassay on a sample taken from the human subject within about 24 hoursafter an actual or suspected injury to the head to measure or detect alevel of cardiac troponin I, wherein the sample is a biological sample;and b) providing a cardioprotective therapy or therapeutic to thesubject if the level of cardiac troponin I in the sample is higher thana reference level of cardiac troponin I. In some embodiments, thecardioprotective therapy optionally can include administering one ormore beta-blockers, diuretics, Angiotensin-Converting Enzyme (ACE)inhibitors, calcium channel blockers, lipid lowering therapies, statins(also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors), nitrates, antiplatelets, anticlotting agents,anticoagulation agents, and the like, such as is known in the art. Thenature, amounts and timing for the administration of suchcardioprotective therapies and therapeutics are well known in the art.

In some embodiments, the sample may be obtained or taken from thesubject within about 0 minutes, within about 1 minute, within about 2minutes, within about 3 minutes, within about 4 minutes, within about 5minutes, within about 6 minutes, within about 7 minutes, within about 8minutes, within about 9 minutes, within about 10 minutes, within about11 minutes, within about 12 minutes, within about 13 minutes, withinabout 14 minutes, within about 15 minutes, within about 20 minutes,within about 30 minutes, within about 1 hour, within about 2 hours,within about 3 hours, within about 4 hours, within about 5 hours, withinabout 6 hours, within about 7 hours, within about 8 hours, within about9 hours, within about 10 hours, within about 11 hours, within about 12hours, within about 13 hours, within about 14 hours, within about 15hours, within about 16 hours, within about 17 hours, within about 18hours, within about 19 hours, within about 20 hours, within about 21hours, within about 22 hours, within about 23 hours or within about 24hours of a suspect injury to the head.

Generally, a reference level of cTnI can also be employed as a benchmarkagainst which to assess results obtained upon assaying a test sample forcTnI. Generally, in making such a comparison, the reference level ofcTnI is obtained by running a particular assay a sufficient number oftimes and under appropriate conditions such that a linkage orassociation of analyte presence, amount or concentration with aparticular stage or endpoint of TBI or with particular indicia can bemade. Typically, the reference level of cTnI is obtained with assays ofreference subjects (or populations of subjects). The cTnI measured caninclude fragments thereof, degradation products thereof, and/orenzymatic cleavage products thereof.

In some embodiments, the method can include obtaining samples from thesubject and contacting the samples with an antibody for cardiac troponinI to allow formation of a complex of the antibody and cardiac troponinI. The method also includes detecting the resulting antibody-cardiactroponin I complex.

The nature of the assay employed in the methods described herein is notcritical and the test can be any assay known in the art such as, forexample, immunoassays, protein immunoprecipitation,immunoelectrophoresis, Western blot, or protein immunostaining, orspectrometry methods, such as high-performance liquid chromatography(HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assaysare described in further detail herein in Sections 8-12. Also, the assaycan be employed in clinical chemistry format such as would be known byone skilled in the art. For example, a clinical chemistry format caninclude an assay that involves one antibody or no antibody. Examples ofanalyzers that can be used for the clinical chemistry format aredescribed in U.S. Patent publication Nos. 2016/0320422 and 2015/0112630.

7. TREATMENT AND MONITORING OF SUBJECTS SUFFERING FROM TRAUMATIC BRAININJURY

The subject identified or assessed in the methods described above ashaving traumatic brain injury, such as mild traumatic brain injury or amoderate, severe, or moderate to severe traumatic brain injury, may betreated or monitored. In some embodiments, the method further includestreating the human subject assessed as having traumatic brain injurywith a traumatic brain injury treatment, such as any treatments known inthe art. For example, treatment of traumatic brain injury can take avariety of forms depending on the severity of the injury to the head.For example, for subjects suffering from mild TBI, the treatment mayinclude one or more of rest, abstaining from physical activities, suchas sports, avoiding light or wearing sunglasses when out in the light,administration of one or more therapeutics (e.g., such as a medicationfor relief of a headache or migraine, anti-nausea medication, etc.).Treatment for patients suffering from a moderate, severe, or moderate tosevere TBI might include administration of one or more appropriatetherapeutics (such as, for example, diuretics, anti-convulsantmedications, medications to sedate and put an individual in adrug-induced coma, or other pharmaceutical or biopharmaceuticalmedications (either known or developed in the future for treatment ofTBI), one or more surgical procedures (such as, for example, removal ofa hematoma, repairing a skull fracture, decompressive craniectomy, etc.)protecting the airway, and one or more therapies (such as, for exampleone or more rehabilitation, physical therapy, occupational therapy,cognitive behavioral therapy, anger management, counseling psychology,etc.). In some embodiments, the method further includes monitoring thehuman subject assessed as having traumatic brain injury (e.g., mild ormoderate, severe, or moderate to severe traumatic). In some embodiments,a subject identified as having traumatic brain injury, such as mildtraumatic brain injury or severe traumatic brain injury, may bemonitored with CT scan or MRI. The treatments employed for mild ormoderate, severe, or moderate to severe TBI described herein can beadministered in connection with one or more cardioprotective therapiesor therapeutics described in Section 6.

8. METHODS FOR MEASURING THE LEVEL OF CTNI

In the methods described above, cTnI levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of cTnI includes contacting thesample with a first specific binding member and second specific bindingmember. In some embodiments the first specific binding member is acapture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of cTnI includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., cTnI-capture antibody), which bindsto an epitope on cTnI or cTnI fragment to form a capture antibody-cTnIantigen complex (e.g., cTnI-capture antibody-cTnI antigen complex), and(2) a detection antibody (e.g., cTnI-detection antibody), which includesa detectable label and binds to an epitope on cTnI that is not bound bythe capture antibody, to form a cTnI antigen-detection antibody complex(e.g., cTnI antigen-cTnI-detection antibody complex), such that acapture antibody-cTnI antigen-detection antibody complex (e.g.,cTnI-capture antibody-cTnI antigen-cTnI-detection antibody complex) isformed, and measuring the amount or concentration of cTnI in the samplebased on the signal generated by the detectable label in the captureantibody-cTnI antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a cTnI antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example the Abbott Laboratoriesinstrument ARCHITECT®, and other core laboratory instruments) other thana point-of-care device may be capable of measuring levels of cTnI in asample at about 0.032 μg/L at 10% CV or lower.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety

9. METHODS FOR MEASURING THE LEVEL OF UCH-L1

In the methods described above, UCH-L1 levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of UCH-L1 includes contactingthe sample with a first specific binding member and second specificbinding member. In some embodiments the first specific binding member isa capture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of UCH-L1 includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., UCH-L1-capture antibody), whichbinds to an epitope on UCH-L1 or UCH-L1 fragment to form a captureantibody-UCH-L1 antigen complex (e.g., UCH-L1-capture antibody-UCH-L1antigen complex), and (2) a detection antibody (e.g., UCH-L1-detectionantibody), which includes a detectable label and binds to an epitope onUCH-L1 that is not bound by the capture antibody, to form a UCH-L1antigen-detection antibody complex (e.g., UCH-L1antigen-UCH-L1-detection antibody complex), such that a captureantibody-UCH-L1 antigen-detection antibody complex (e.g., UCH-L1-captureantibody-UCH-L1 antigen-UCH-L1-detection antibody complex) is formed,and measuring the amount or concentration of UCH-L1 in the sample basedon the signal generated by the detectable label in the captureantibody-UCH-L1 antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a UCH-L1 antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example the Abbott Laboratoriesinstrument ARCHITECT®, and other core laboratory instruments) other thana point-of-care device may be capable of measuring levels of UCH-L1 in asample higher or greater than 25,000 pg/mL.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety.

10. METHODS FOR MEASURING THE LEVEL OF GFAP

In the methods described above, GFAP levels can be measured by anymeans, such as antibody dependent methods, such as immunoassays, proteinimmunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGEand Western blot analysis, or protein immunostaining, electrophoresisanalysis, a protein assay, a competitive binding assay, a functionalprotein assay, or chromatography or spectrometry methods, such ashigh-performance liquid chromatography (HPLC) or liquidchromatography-mass spectrometry (LC/MS). Also, the assay can beemployed in clinical chemistry format such as would be known by oneskilled in the art.

In some embodiments, measuring the level of GFAP includes contacting thesample with a first specific binding member and second specific bindingmember. In some embodiments the first specific binding member is acapture antibody and the second specific binding member is a detectionantibody. In some embodiments, measuring the level of GFAP includescontacting the sample, either simultaneously or sequentially, in anyorder: (1) a capture antibody (e.g., GFAP-capture antibody), which bindsto an epitope on GFAP or GFAP fragment to form a capture antibody-GFAPantigen complex (e.g., GFAP-capture antibody-GFAP antigen complex), and(2) a detection antibody (e.g., GFAP-detection antibody), which includesa detectable label and binds to an epitope on GFAP that is not bound bythe capture antibody, to form a GFAP antigen-detection antibody complex(e.g., GFAP antigen-GFAP-detection antibody complex), such that acapture antibody-GFAP antigen-detection antibody complex (e.g.,GFAP-capture antibody-GFAP antigen-GFAP-detection antibody complex) isformed, and measuring the amount or concentration of GFAP in the samplebased on the signal generated by the detectable label in the captureantibody-GFAP antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized ona solid support. In some embodiments, the second specific binding memberis immobilized on a solid support. In some embodiments, the firstspecific binding member is a GFAP antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample canbe from about 1 to about 25 microliters, about 1 to about 24microliters, about 1 to about 23 microliters, about 1 to about 22microliters, about 1 to about 21 microliters, about 1 to about 20microliters, about 1 to about 18 microliters, about 1 to about 17microliters, about 1 to about 16 microliters, about 15 microliters orabout 1 microliter, about 2 microliters, about 3 microliters, about 4microliters, about 5 microliters, about 6 microliters, about 7microliters, about 8 microliters, about 9 microliters, about 10microliters, about 11 microliters, about 12 microliters, about 13microliters, about 14 microliters, about 15 microliters, about 16microliters, about 17 microliters, about 18 microliters, about 19microliters, about 20 microliters, about 21 microliters, about 22microliters, about 23 microliters, about 24 microliters or about 25microliters. In some embodiments, the sample is from about 1 to about150 microliters or less or from about 1 to about 25 microliters or less.

Some instruments (such as, for example the Abbott Laboratoriesinstrument ARCHITECT®, and other core laboratory instruments) other thana point-of-care device may be capable of measuring levels of GFAP in asample higher or greater than 25,000 pg/mL.

Other methods of detection include the use of or can be adapted for useon a nanopore device or nanowell device. Examples of nanopore devicesare described in International Patent Publication No. WO 2016/161402,which is hereby incorporated by reference in its entirety. Examples ofnanowell device are described in International Patent Publication No. WO2016/161400, which is hereby incorporated by reference in its entirety.

11. ANTIBODIES

The methods described herein may use an isolated antibody thatspecifically binds to the cardiac troponin I (cTnI) and/or isolatedantibody that specifically binds to the early biomarker which is notcTnI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glialfibrillary acidic protein (GFAP), or a combination thereof

a. Cardiac Troponin I Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to cardiac troponin I, such as, for example, humancardiac troponin I (or fragments thereof), referred to as “cardiactroponin I antibody.” Cardiac troponin I antibodies can be used toassess the cardiac troponin I status as a measure of traumatic braininjury, detect the presence of cardiac troponin I in a biologicalsample, quantify the amount of cardiac troponin I present in abiological sample, or detect the presence of and quantify the amount ofcardiac troponin I in a biological sample.

(1) Human Cardiac Troponin I (cTnI)

Human cardiac troponin I (cTnI) along with troponin T (TnT) and troponinC (TnC), are the 3 subunits that form the troponin complex of the thinfilaments of striated muscle. Cardiac troponin I is the inhibitorysubunit; blocking actin-myosin interactions and thereby mediatingstriated muscle relaxation. The cTnI subfamily contains three genes:cTnI-skeletal-fast-twitch, cTnI-skeletal-slow-twitch, and cTnI-cardiac.This gene encodes the cTnI-cardiac protein and is exclusively expressedin cardiac muscle tissues.

Human cardiac troponin I may have the following amino acid sequence:

(SEQ ID NO: 1) MADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAKQELEREAEERRGEKGRALSTRCQPLELAGLGFAELQDLCRQLHARVDKVDEERYDIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVRISADAMMQALLGARAKESLDLRAHLKQVKKEDTEKENREVGDWRKNIDALSG MEGRKKKFES.

The human cardiac troponin I may be a fragment or variant of SEQ IDNO: 1. The fragment of cardiac troponin I may be between 5 and 210 aminoacids, between 10 and 210 amino acids, between 50 and 210 amino acids,between 60 and 210 amino acids, between 65 and 210 amino acids, between100 and 210 amino acids, between 150 and 210 amino acids, between 100and 210 amino acids, or between 175 and 210 amino acids in length. Thefragment may comprise a contiguous number of amino acids from SEQ ID NO:1.

(2) Cardiac Troponin I-Recognizing Antibody

The antibody is an antibody that binds to cardiac troponin I, a fragmentthereof, an epitope of cardiac troponin I, or a variant thereof. Theantibody may be a fragment of the anti-cardiac troponin I antibody or avariant or a derivative thereof. The antibody may be a polyclonal ormonoclonal antibody. The antibody may be a chimeric antibody, a singlechain antibody, an affinity matured antibody, a human antibody, ahumanized antibody, a fully human antibody or an antibody fragment, suchas a Fab fragment, or a mixture thereof. Antibody fragments orderivatives may comprise F(ab′)₂, Fv or scFv fragments. The antibodyderivatives can be produced by peptidomimetics. Further, techniquesdescribed for the production of single chain antibodies can be adaptedto produce single chain antibodies.

The anti-cardiac troponin I antibodies may be a chimeric anti-cardiactroponin I or humanized anti-cardiac troponin I antibody. In oneembodiment, both the humanized antibody and chimeric antibody aremonovalent. In one embodiment, both the humanized antibody and chimericantibody comprise a single Fab region linked to an Fc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-cardiac troponin I antibodies. Forexample, libraries of naïve human single chain variable fragments (scFv)may be used to select human anti-cardiac troponin I antibodies.Transgenic animals may be used to express human antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

i. Epitope

The antibody may immunospecifically bind to human cardiac troponin I(SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibodymay immunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

(3) Anti-Cardiac Troponin I Antibodies

Anti-cardiac troponin I antibodies may be generated using the techniquesdescribed above as well as using routine techniques known in the art. Insome embodiments, the anti-cardiac troponin I antibody may be anunconjugated cardiac troponin I antibody, such as cardiac troponin Iantibodies available from Abcam (such as Anti-Cardiac troponin Iantibody (ab47003)), Thermofisher (such as cardiac troponin I monoclonalantibody (12F10), cardiac troponin I polyclonal antibody, cardiactroponin I antibody (1HCLC), ABFINITY™ rabbit oligoclonal, cardiactroponin I antibody (1H11L19), ABFINITY™ rabbit monoclonal), Santa Cruz(such as cardiac troponin I antibody (C-4) (Catalog number sc-133117),cardiac troponin I antibody (4) (Catalog number sc-130351), cardiactroponin I antibody (12) (Catalog number sc-130350), cardiac troponin Iantibody (H-170) (Catalog number sc-15368), cardiac troponin I antibody(C-19) (Catalog number sc-8118), cardiac troponin I-C antibody (G-11)(Catalog number sc-376662), cardiac troponin I-C antibody (M46) (Catalognumber sc-52277), cardiac troponin I-C antibody (10B11) (Catalog numbersc-52266) and hytest (Monoclonal mouse anti-cardiac cardiac troponin I(catalog number 4T21).

b. UCH-L1 Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to ubiquitin carboxy-terminal hydrolase L1 (“UCH-L1”)(or fragments thereof), referred to as “UCH-L1 antibody.” The UCH-L1antibodies can be used to assess the UCH-L1 status as a measure oftraumatic brain injury, detect the presence of UCH-L1 in a sample,quantify the amount of UCH-L1 present in a sample, or detect thepresence of and quantify the amount of UCH-L1 in a sample.

(1) Ubiquitin Carboxy-Terminal Hydrolase L1 (UCH-L1)

Ubiquitin carboxy-terminal hydrolase L1 (“UCH-L1”), which is also knownas “ubiquitin C-terminal hydrolase,” is a deubiquitinating enzyme.UCH-L1 is a member of a gene family whose products hydrolyze smallC-terminal adducts of ubiquitin to generate the ubiquitin monomer.Expression of UCH-L1 is highly specific to neurons and to cells of thediffuse neuroendocrine system and their tumors. It is abundantly presentin all neurons (accounts for 1-2% of total brain protein), expressedspecifically in neurons and testis/ovary. The catalytic triad of UCH-L1contains a cysteine at position 90, an aspartate at position 176, and ahistidine at position 161 that are responsible for its hydrolaseactivity.

Human UCH-L1 may have the following amino acid sequence:

(SEQ ID NO: 2) MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALCKAA.

The human UCH-L1 may be a fragment or variant of SEQ ID NO: 2. Thefragment of UCH-L1 may be between 5 and 225 amino acids, between 10 and225 amino acids, between 50 and 225 amino acids, between 60 and 225amino acids, between 65 and 225 amino acids, between 100 and 225 aminoacids, between 150 and 225 amino acids, between 100 and 175 amino acids,or between 175 and 225 amino acids in length. The fragment may comprisea contiguous number of amino acids from SEQ ID NO: 2.

(2) UCH-L1-Recognizing Antibody

The antibody is an antibody that binds to UCH-L1, a fragment thereof, anepitope of UCH-L1, or a variant thereof. The antibody may be a fragmentof the anti-UCH-L1 antibody or a variant or a derivative thereof. Theantibody may be a polyclonal or monoclonal antibody. The antibody may bea chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, a fully human antibodyor an antibody fragment, such as a Fab fragment, or a mixture thereof.Antibody fragments or derivatives may comprise F(ab′)₂, Fv or scFvfragments. The antibody derivatives can be produced by peptidomimetics.Further, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies.

The anti-UCH-L1 antibodies may be a chimeric anti-UCH-L1 or humanizedanti-UCH-L1 antibody. In one embodiment, both the humanized antibody andchimeric antibody are monovalent. In one embodiment, both the humanizedantibody and chimeric antibody comprise a single Fab region linked to anFc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-UCH-L1 antibodies. For example, librariesof naïve human single chain variable fragments (scFv) may be used toselect human anti-UCH-L1 antibodies. Transgenic animals may be used toexpress human antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

i. Epitope

The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 2), afragment thereof, or a variant thereof. The antibody mayimmunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

(3) Anti-UCH-L1 Antibodies

Anti-UCH-L1 antibodies may be generated using the techniques describedabove as well as using routine techniques known in the art. In someembodiments, the anti-UCH-L1 antibody may be an unconjugated UCH-L1antibody, such as UCH-L1 antibodies available from United StateBiological (Catalog Number: 031320), Cell Signaling Technology (CatalogNumber: 3524), Sigma-Aldrich (Catalog Number: HPA005993), Santa CruzBiotechnology, Inc. (Catalog Numbers: sc-58593 or sc-58594), R&D Systems(Catalog Number: MAB6007), Novus Biologicals (Catalog Number:NB600-1160), Biorbyt (Catalog Number: orb33715), Enzo Life Sciences,Inc. (Catalog Number: ADI-905-520-1), Bio-Rad (Catalog Number:VMA00004), BioVision (Catalog Number: 6130-50), Abcam (Catalog Numbers:ab75275 or ab104938), Invitrogen Antibodies (Catalog Numbers: 480012),ThermoFisher Scientific (Catalog Numbers: MA1-46079, MA5-17235,MA1-90008, or MA1-83428), EMD Millipore (Catalog Number: MABN48), orSino Biological Inc. (Catalog Number: 50690-R011). The anti-UCH-L1antibody may be conjugated to a fluorophore, such as conjugated UCH-L1antibodies available from BioVision (Catalog Number: 6960-25) or AvivaSystems Biology (Cat. Nos. OAAF01904-FITC).

c. GFAP Antibodies

The methods described herein may use an isolated antibody thatspecifically binds to Glial fibrillary acidic protein (“GFAP”) (orfragments thereof), referred to as “GFAP antibody.” The GFAP antibodiescan be used to assess the GFAP status as a measure of traumatic braininjury, detect the presence of GFAP in a sample, quantify the amount ofGFAP present in a sample, or detect the presence of and quantify theamount of GFAP in a sample.

(1) Glial Fibrillary Acidic Protein (GFAP)

Glial fibrillary acidic protein (GFAP) is a 50 kDa intracytoplasmicfilamentous protein that constitutes a portion of the cytoskeleton inastrocytes, and it has proved to be the most specific marker for cellsof astrocytic origin. GFAP protein is encoded by the GFAP gene inhumans. GFAP is the principal intermediate filament of matureastrocytes. In the central rod domain of the molecule, GFAP sharesconsiderable structural homology with the other intermediate filaments.GFAP is involved in astrocyte motility and shape by providing structuralstability to astrocytic processes. Glial fibrillary acidic protein andits breakdown products (GFAP-BDP) are brain-specific proteins releasedinto the blood as part of the pathophysiological response aftertraumatic brain injury (TBI). Following injury to the human CNS causedby trauma, genetic disorders, or chemicals, astrocytes proliferate andshow extensive hypertrophy of the cell body and processes, and GFAP ismarkedly upregulated. In contrast, with increasing astrocyte malignancy,there is a progressive loss of GFAP production. GFAP can also bedetected in Schwann cells, enteric glia cells, salivary gland neoplasms,metastasizing renal carcinomas, epiglottic cartilage, pituicytes,immature oligodendrocytes, papillary meningiomas, and myoepithelialcells of the breast.

Human GFAP may have the following amino acid sequence:

(SEQ ID NO: 3) MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.

The human GFAP may be a fragment or variant of SEQ ID NO: 3. Thefragment of GFAP may be between 5 and 400 amino acids, between 10 and400 amino acids, between 50 and 400 amino acids, between 60 and 400amino acids, between 65 and 400 amino acids, between 100 and 400 aminoacids, between 150 and 400 amino acids, between 100 and 300 amino acids,or between 200 and 300 amino acids in length. The fragment may comprisea contiguous number of amino acids from SEQ ID NO: 3. The human GFAPfragment or variant of SEQ ID NO: 3 may be a GFAP breakdown product(BDP). The GFAP BDP may be 38 kDa, 42 kDa (fainter 41 kDa), 47 kDa(fainter 45 kDa); 25 kDa (fainter 23 kDa); 19 kDa, or 20 kDa.

(2) GFAP-Recognizing Antibody

The antibody is an antibody that binds to GFAP, a fragment thereof, anepitope of GFAP, or a variant thereof. The antibody may be a fragment ofthe anti-GFAP antibody or a variant or a derivative thereof. Theantibody may be a polyclonal or monoclonal antibody. The antibody may bea chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, a fully human antibodyor an antibody fragment, such as a Fab fragment, or a mixture thereof.Antibody fragments or derivatives may comprise F(ab′)2, Fv or scFvfragments. The antibody derivatives can be produced by peptidomimetics.Further, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies.

The anti-GFAP antibodies may be a chimeric anti-GFAP or humanizedanti-GFAP antibody. In one embodiment, both the humanized antibody andchimeric antibody are monovalent. In one embodiment, both the humanizedantibody and chimeric antibody comprise a single Fab region linked to anFc region.

Human antibodies may be derived from phage-display technology or fromtransgenic mice that express human immunoglobulin genes. The humanantibody may be generated as a result of a human in vivo immune responseand isolated. See, for example, Funaro et al., BMC Biotechnology,2008(8):85. Therefore, the antibody may be a product of the human andnot animal repertoire. Because it is of human origin, the risks ofreactivity against self-antigens may be minimized. Alternatively,standard yeast display libraries and display technologies may be used toselect and isolate human anti-GFAP antibodies. For example, libraries ofnaïve human single chain variable fragments (scFv) may be used to selecthuman anti-GFAP antibodies. Transgenic animals may be used to expresshuman antibodies.

Humanized antibodies may be antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

The antibody is distinguishable from known antibodies in that itpossesses different biological function(s) than those known in the art.

i. Epitope

The antibody may immunospecifically bind to GFAP (SEQ ID NO: 3), afragment thereof, or a variant thereof. The antibody mayimmunospecifically recognize and bind at least three amino acids, atleast four amino acids, at least five amino acids, at least six aminoacids, at least seven amino acids, at least eight amino acids, at leastnine amino acids, or at least ten amino acids within an epitope region.The antibody may immunospecifically recognize and bind to an epitopethat has at least three contiguous amino acids, at least four contiguousamino acids, at least five contiguous amino acids, at least sixcontiguous amino acids, at least seven contiguous amino acids, at leasteight contiguous amino acids, at least nine contiguous amino acids, orat least ten contiguous amino acids of an epitope region.

(3) Anti-GFAP Antibodies

Anti-GFAP antibodies may be generated using the techniques describedabove as well as using routine techniques known in the art. In someembodiments, the anti-GFAP antibody may be an unconjugated GFAPantibody, such as GFAP antibodies available from Dako (Catalog Number:M0761), ThermoFisher Scientific (Catalog Numbers: MA5-12023, A-21282,13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377,MA1-06701, or MA1-20035), AbCam (Catalog Numbers: ab10062, ab4648,ab68428, ab33922, ab207165, ab190288, ab115898, or ab21837), EMDMillipore (Catalog Numbers: FCMAB257P, MAB360, MAB3402, 04-1031,04-1062, MAB5628), Santa Cruz (Catalog Numbers: sc-166481, sc-166458,sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343, orsc-33673), Sigma-Aldrich (Catalog Numbers: G3893 or G6171) or SinoBiological Inc. (Catalog Number: 100140-R012-50). The anti-GFAP antibodymay be conjugated to a fluorophore, such as conjugated GFAP antibodiesavailable from ThermoFisher Scientific (Catalog Numbers: A-21295 orA-21294), EMD Millipore (Catalog Numbers: MAB3402X, MAB3402B, MAB3402B,or MAB3402C3) or AbCam (Catalog Numbers: ab49874 or ab194325).

d. Antibody Preparation/Production

Antibodies may be prepared by any of a variety of techniques, includingthose well known to those skilled in the art. In general, antibodies canbe produced by cell culture techniques, including the generation ofmonoclonal antibodies via conventional techniques, or via transfectionof antibody genes, heavy chains, and/or light chains into suitablebacterial or mammalian cell hosts, in order to allow for the productionof antibodies, wherein the antibodies may be recombinant. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies in either prokaryoticor eukaryotic host cells, expression of antibodies in eukaryotic cellsis preferable, and most preferable in mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesinclude Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0myeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody. Recombinant DNA technology may also be used to remove some, orall, of the DNA encoding either or both of the light and heavy chainsthat is not necessary for binding to the antigens of interest. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies. In addition, bifunctional antibodies maybe produced in which one heavy and one light chain are an antibody(i.e., binds an analyte, e.g., human troponin I, UCH-L1, or GFAP) andthe other heavy and light chain are specific for an antigen other thanthe analyte by crosslinking an antibody to a second antibody by standardchemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the method of synthesizing a recombinant antibodymay be by culturing a host cell in a suitable culture medium until arecombinant antibody is synthesized. The method can further compriseisolating the recombinant antibody from the culture medium.

Methods of preparing monoclonal antibodies involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Such cell lines may be produced from spleen cells obtainedfrom an immunized animal. The animal may be immunized with the analyte(e.g., cardiac troponin I, UCH-L1, or GFAP) or a fragment and/or variantthereof. The peptide used to immunize the animal may comprise aminoacids encoding human Fc, for example the fragment crystallizable regionor tail region of human antibody. The spleen cells may then beimmortalized by, for example, fusion with a myeloma cell fusion partner.A variety of fusion techniques may be employed. For example, the spleencells and myeloma cells may be combined with a nonionic detergent for afew minutes and then plated at low density on a selective medium thatsupports that growth of hybrid cells, but not myeloma cells. One suchtechnique uses hypoxanthine, aminopterin, thymidine (HAT) selection.Another technique includes electrofusion. After a sufficient time,usually about 1 to 2 weeks, colonies of hybrids are observed. Singlecolonies are selected and their culture supernatants tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity may be used.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. Affinity chromatography is an example ofa method that can be used in a process to purify the antibodies.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment, which comprises bothantigen-binding sites.

The Fv fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecules. TheFv fragment may be derived using recombinant techniques. The Fv fragmentincludes a non-covalent VH::VL heterodimer including an antigen-bindingsite that retains much of the antigen recognition and bindingcapabilities of the native antibody molecule.

The antibody, antibody fragment, or derivative may comprise a heavychain and a light chain complementarity determining region (“CDR”) set,respectively interposed between a heavy chain and a light chainframework (“FR”) set which provide support to the CDRs and define thespatial relationship of the CDRs relative to each other. The CDR set maycontain three hypervariable regions of a heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g.,as available from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al.(1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134 (1994)).

An affinity matured antibody may be produced by any one of a number ofprocedures that are known in the art. For example, see Marks et al.,BioTechnology, 10: 779-783 (1992) describes affinity maturation by VHand VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

Antibody variants can also be prepared using delivering a polynucleotideencoding an antibody to a suitable host such as to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. These methods are known inthe art and are described for example in U.S. Pat. Nos. 5,827,690;5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

Antibody variants also can be prepared by delivering a polynucleotide toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. For example, Cramer et al. (1999) Curr. Top. Microbiol.Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 andreferences cited therein. Antibody variants have also been produced inlarge amounts from transgenic plant seeds including antibody fragments,such as single chain antibodies (scFv's), including tobacco seeds andpotato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol.38:101-109 and reference cited therein. Thus, antibodies can also beproduced using transgenic plants, according to known methods.

Antibody derivatives can be produced, for example, by adding exogenoussequences to modify immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic. Generally, part or all of the non-humanor human CDR sequences are maintained while the non-human sequences ofthe variable and constant regions are replaced with human or other aminoacids.

Small antibody fragments may be diabodies having two antigen-bindingsites, wherein fragments comprise a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (VH VL). See for example, EP 404,097; WO 93/11161; and Hollingeret al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using alinker that is too short to allow pairing between the two domains on thesame chain, the domains are forced to pair with the complementarydomains of another chain and create two antigen-binding sites. See also,U.S. Pat. No. 6,632,926 to Chen et al. which is hereby incorporated byreference in its entirety and discloses antibody variants that have oneor more amino acids inserted into a hypervariable region of the parentantibody and a binding affinity for a target antigen which is at leastabout two fold stronger than the binding affinity of the parent antibodyfor the antigen.

The antibody may be a linear antibody. The procedure for making a linearantibody is known in the art and described in Zapata et al., (1995)Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pairof tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The antibodies may be recovered and purified from recombinant cellcultures by known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

It may be useful to detectably label the antibody. Methods forconjugating antibodies to these agents are known in the art. For thepurpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.They can be linked to a cytokine, to a ligand, to another antibody.Suitable agents for coupling to antibodies to achieve an anti-tumoreffect include cytokines, such as interleukin 2 (IL-2) and TumorNecrosis Factor (TNF); photosensitizers, for use in photodynamictherapy, including aluminum (III) phthalocyanine tetrasulfonate,hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131(1314 yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re);antibiotics, such as doxorubicin, adriamycin, daunorubicin,methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial,plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxinA, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylatedricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinesecobra (naj a naj a atra), and gelonin (a plant toxin); ribosomeinactivating proteins from plants, bacteria and fungi, such asrestrictocin (a ribosome inactivating protein produced by Aspergillusrestrictus), saporin (a ribosome inactivating protein from Saponariaofficinalis), and RNase; tyrosine kinase inhibitors; ly207702 (adifluorinated purine nucleoside); liposomes containing anti cysticagents (e.g., antisense oligonucleotides, plasmids which encode fortoxins, methotrexate, etc.); and other antibodies or antibody fragments,such as F(ab).

Antibody production via the use of hybridoma technology, the selectedlymphocyte antibody method (SLAM), transgenic animals, and recombinantantibody libraries is described in more detail below.

(1) Anti-Analyte Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is alsonoted that the term “monoclonal antibody” as used herein is not limitedto antibodies produced through hybridoma technology. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

Methods of generating monoclonal antibodies as well as antibodiesproduced by the method may comprise culturing a hybridoma cell secretingan antibody of the disclosure wherein, preferably, the hybridoma isgenerated by fusing splenocytes isolated from an animal, e.g., a rat ora mouse, immunized with the analyte (e.g., cardiac troponin I, UCH-L1,or GFAP) with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the disclosure. Briefly, rats can be immunizedwith an analyte (e.g., cardiac troponin I, UCH-L1, or GFAP) antigen. Ina preferred embodiment, the analyte (e.g., cardiac troponin I, UCH-L1,or GFAP) antigen is administered with an adjuvant to stimulate theimmune response. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks; however, a single administration of thepolypeptide may also be used.

After immunization of an animal with an analyte (e.g., cardiac troponinI, UCH-L1, or GFAP) antigen, antibodies and/or antibody-producing cellsmay be obtained from the animal. An anti-analyte (e.g., cardiac troponinI, UCH-L1, or GFAP) antibody-containing serum is obtained from theanimal by bleeding or sacrificing the animal. The serum may be used asit is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-analyte (e.g., cardiac troponin I,UCH-L1, or GFAP) antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen analyte (e.g., cardiac troponin I, UCH-L1, or GFAP) are detectedin the rat serum, the rat spleen is harvested and splenocytes isolated.The splenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example, cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC, Manassas, Va., US). Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding the analyte (e.g., cardiac troponin I,UCH-L1, or GFAP). Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing rats with positive hybridomaclones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using the analyte (e.g., cardiactroponin I, UCH-L1, or GFAP), or a portion thereof, or a cell expressingthe analyte (e.g., cardiac troponin I, UCH-L1, or GFAP). In a preferredembodiment, the initial screening is performed using an enzyme-linkedimmunosorbent assay (ELISA) or a radioimmunoassay (RIA), preferably anELISA. An example of ELISA screening is provided in PCT Publication No.WO 00/37504.

Anti-analyte (e.g., cardiac troponin I, UCH-L1, or GFAP)antibody-producing hybridomas are selected, cloned, and further screenedfor desirable characteristics, including robust hybridoma growth, highantibody production, and desirable antibody characteristics. Hybridomasmay be cultured and expanded in vivo in syngeneic animals, in animalsthat lack an immune system, e.g., nude mice, or in cell culture invitro. Methods of selecting, cloning and expanding hybridomas are wellknown to those of ordinary skill in the art.

In a preferred embodiment, hybridomas are rat hybridomas. In anotherembodiment, hybridomas are produced in a non-human, non-rat species suchas mice, sheep, pigs, goats, cattle, or horses. In yet another preferredembodiment, the hybridomas are human hybridomas, in which a humannon-secretory myeloma is fused with a human cell expressing ananti-analyte (e.g., cardiac troponin I, UCH-L1, or GFAP) antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of thedisclosure may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce two identical Fabfragments) or pepsin (to produce an F(ab′)₂ fragment). A F(ab′)₂fragment of an IgG molecule retains the two antigen-binding sites of thelarger (“parent”) IgG molecule, including both light chains (containingthe variable light chain and constant light chain regions), the CH1domains of the heavy chains, and a disulfide-forming hinge region of theparent IgG molecule. Accordingly, an F(ab′)₂ fragment is still capableof crosslinking antigen molecules like the parent IgG molecule.

(2) Anti-Analyte Monoclonal Antibodies Using SLAM

In another aspect of the disclosure, recombinant antibodies aregenerated from single, isolated lymphocytes using a procedure referredto in the art as the selected lymphocyte antibody method (SLAM), asdescribed in U.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551;and Babcook et al., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). Inthis method, single cells secreting antibodies of interest, e.g.,lymphocytes derived from any one of the immunized animals are screenedusing an antigen-specific hemolytic plaque assay, wherein the antigenanalyte (e.g., cardiac troponin I, UCH-L1, or GFAP), a subunit of theanalyte (e.g., cardiac troponin I, UCH-L1, or GFAP), or a fragmentthereof, is coupled to sheep red blood cells using a linker, such asbiotin, and used to identify single cells that secrete antibodies withspecificity for the analyte (e.g., cardiac troponin I, UCH-L1, or GFAP).Following identification of antibody-secreting cells of interest, heavy-and light-chain variable region cDNAs are rescued from the cells byreverse transcriptase-PCR (RT-PCR) and these variable regions can thenbe expressed, in the context of appropriate immunoglobulin constantregions (e.g., human constant regions), in mammalian host cells, such asCOS or CHO cells. The host cells transfected with the amplifiedimmunoglobulin sequences, derived from in vivo selected lymphocytes, canthen undergo further analysis and selection in vitro, for example, bypanning the transfected cells to isolate cells expressing antibodies tothe analyte (e.g., cardiac troponin I, UCH-L1, or GFAP). The amplifiedimmunoglobulin sequences further can be manipulated in vitro, such as byin vitro affinity maturation method. See, for example, PCT PublicationNo. WO 97/29131 and PCT Publication No. WO 00/56772.

(3) Anti-Analyte Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the disclosure, antibodies are produced byimmunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an analyte (e.g., cardiac troponin I, UCH-L1,or GFAP) antigen. In an embodiment, the non-human animal is a XENOMOUSE®transgenic mouse, an engineered mouse strain that comprises largefragments of the human immunoglobulin loci and is deficient in mouseantibody production. See, e.g., Green et al., Nature Genetics, 7: 13-21(1994) and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209;6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT PublicationNos. WO 91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO98/24893; WO 98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and WO00/37504. The XENOMOUSE® transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman monoclonal antibodies. The XENOMOUSE® transgenic mouse containsapproximately 80% of the human antibody repertoire through introductionof megabase sized, germline configuration YAC fragments of the humanheavy chain loci and x light chain loci. See Mendez et al., NatureGenetics, 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are hereby incorporated byreference.

(4) Anti-Analyte Monoclonal Antibodies Using Recombinant AntibodyLibraries

In vitro methods also can be used to make the antibodies of thedisclosure, wherein an antibody library is screened to identify anantibody having the desired analyte (e.g., cardiac troponin I, UCH-L1,or GFAP)-binding specificity. Methods for such screening of recombinantantibody libraries are well known in the art and include methodsdescribed in, for example, U.S. Pat. No. 5,223,409 (Ladner et al.); PCTPublication No. WO 92/18619 (Kang et al.); PCT Publication No. WO91/17271 (Dower et al.); PCT Publication No. WO 92/20791 (Winter etal.); PCT Publication No. WO 92/15679 (Markland et al.); PCT PublicationNo. WO 93/01288 (Breitling et al.); PCT Publication No. WO 92/01047(McCafferty et al.); PCT Publication No. WO 92/09690 (Garrard et al.);Fuchs et al., Bio/Technology, 9: 1369-1372 (1991); Hay et al., Hum.Antibod. Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990);Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol.Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628(1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992);Garrard et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al.,Nucl. Acids Res., 19: 4133-4137 (1991); Barbas et al., Proc. Natl. Acad.Sci. USA, 88: 7978-7982 (1991); U.S. Patent Application Publication No.2003/0186374; and PCT Publication No. WO 97/29131, the contents of eachof which are incorporated herein by reference.

The recombinant antibody library may be from a subject immunized withthe analyte (e.g., cardiac troponin I, UCH-L1, or GFAP), or a portion ofthe analyte (e.g., cardiac troponin I, UCH-L1, or GFAP). Alternatively,the recombinant antibody library may be from a naive subject, i.e., onewho has not been immunized with the analyte (e.g., cardiac troponin I,UCH-L1, or GFAP), such as a human antibody library from a human subjectwho has not been immunized with human analyte (e.g., cardiac troponin I,UCH-L1, or GFAP). Antibodies of the disclosure are selected by screeningthe recombinant antibody library with the peptide comprising humananalyte (e.g., cardiac troponin I, UCH-L1, or GFAP) to thereby selectthose antibodies that recognize the analyte (e.g., cardiac troponin I,UCH-L1, or GFAP). Methods for conducting such screening and selectionare well known in the art, such as described in the references in thepreceding paragraph. To select antibodies of the disclosure havingparticular binding affinities for the analyte (e.g., cardiac troponin I,UCH-L1, or GFAP), such as those that dissociate from human analyte(e.g., cardiac troponin I, UCH-L1, or GFAP) I with a particular K_(off)rate constant, the art-known method of surface plasmon resonance can beused to select antibodies having the desired K_(off) rate constant. Toselect antibodies of the disclosure having a particular neutralizingactivity for the analyte (e.g., cardiac troponin I, UCH-L1, or GFAP),such as those with a particular IC₅₀, standard methods known in the artfor assessing the inhibition of the analyte (e.g., cardiac troponin I,UCH-L1, or GFAP) activity may be used.

In one aspect, the disclosure pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human analyte (e.g., cardiactroponin I, UCH-L1, or GFAP). Preferably, the antibody is a neutralizingantibody. In various embodiments, the antibody is a recombinant antibodyor a monoclonal antibody.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. Such phage can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Phageexpressing an antigen binding domain that binds the antigen of interestcan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv, or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies include those disclosed in Brinkmannet al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al.,Advances in Immunology, 57: 191-280 (1994); PCT Publication No. WO92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743; and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al.,Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al.,Science, 240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the disclosure. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98/31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology is PROfusion display technology.

In another approach, the antibodies can also be generated using yeastdisplay methods known in the art. In yeast display methods, geneticmethods are used to tether antibody domains to the yeast cell wall anddisplay them on the surface of yeast. In particular, such yeast can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Examples ofyeast display methods that can be used to make the antibodies includethose disclosed in U.S. Pat. No. 6,699,658 (Wittrup et al.) incorporatedherein by reference.

e. Production of Recombinant Analyte Antibodies

Antibodies may be produced by any of a number of techniques known in theart. For example, expression from host cells, wherein expressionvector(s) encoding the heavy and light chains is (are) transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection, and the like. Although it ispossible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the disclosure include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0myeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure may be performed. For example, it maybe desirable to transfect a host cell with DNA encoding functionalfragments of either the light chain and/or the heavy chain of anantibody of this disclosure. Recombinant DNA technology may also be usedto remove some, or all, of the DNA encoding either or both of the lightand heavy chains that is not necessary for binding to the antigens ofinterest. The molecules expressed from such truncated DNA molecules arealso encompassed by the antibodies of the disclosure. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the disclosure (i.e., binds human analyte(e.g., cardiac troponin I, UCH-L1, or GFAP)) and the other heavy andlight chain are specific for an antigen other than human analyte (e.g.,cardiac troponin I, UCH-L1, or GFAP) by crosslinking an antibody of thedisclosure to a second antibody by standard chemical crosslinkingmethods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the disclosure, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells, and recover the antibody from the culturemedium. Still further, the disclosure provides a method of synthesizinga recombinant antibody of the disclosure by culturing a host cell of thedisclosure in a suitable culture medium until a recombinant antibody ofthe disclosure is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

(1) Humanized Antibody

The humanized antibody may be an antibody or a variant, derivative,analog or portion thereof which immunospecifically binds to an antigenof interest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. The humanized antibody may befrom a non-human species antibody that binds the desired antigen havingone or more complementarity determining regions (CDRs) from thenon-human species and framework regions from a human immunoglobulinmolecule.

As used herein, the term “substantially” in the context of a CDR refersto a CDR having an amino acid sequence at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′,F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. According to one aspect, ahumanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or of a heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3, and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In one embodiment, suchmutations, however, will not be extensive. Usually, at least 90%, atleast 95%, at least 98%, or at least 99% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences.As used herein, the term “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence. As used herein, theterm “consensus immunoglobulin sequence” refers to the sequence formedfrom the most frequently occurring amino acids (or nucleotides) in afamily of related immunoglobulin sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In afamily of immunoglobulins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

The humanized antibody may be designed to minimize unwantedimmunological response toward rodent anti-human antibodies, which limitsthe duration and effectiveness of therapeutic applications of thosemoieties in human recipients. The humanized antibody may have one ormore amino acid residues introduced into it from a source that isnon-human. These non-human residues are often referred to as “import”residues, which are typically taken from a variable domain. Humanizationmay be performed by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. For example, see U.S.Pat. No. 4,816,567, the contents of which are herein incorporated byreference. The humanized antibody may be a human antibody in which somehypervariable region residues, and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.Humanization or engineering of antibodies of the present disclosure canbe performed using any known method, such as but not limited to thosedescribed in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483;5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023;6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

The humanized antibody may retain high affinity for the analyte (e.g.,cardiac troponin I, UCH-L1, or GFAP) and other favorable biologicalproperties. The humanized antibody may be prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three-dimensional models of the parental and humanizedsequences. Three-dimensional immunoglobulin models are commonlyavailable. Computer programs are available that illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristics, such as increased affinity for the analyte (e.g.,cardiac troponin I, UCH-L1, or GFAP), is achieved. In general, thehypervariable region residues may be directly and most substantiallyinvolved in influencing antigen binding.

As an alternative to humanization, human antibodies (also referred toherein as “fully human antibodies”) can be generated. For example, it ispossible to isolate human antibodies from libraries via PROfusion and/oryeast related technologies. It is also possible to produce transgenicanimals (e.g., mice that are capable, upon immunization, of producing afull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. The humanized or fully humanantibodies may be prepared according to the methods described in U.S.Pat. Nos. 5,770,429; 5,833,985; 5,837,243; 5,922,845; 6,017,517;6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;6,682,928; and 6,984,720, the contents each of which are hereinincorporated by reference.

12. VARIATIONS ON METHODS

The disclosed methods of determining the presence or amount of analytesof interest (e.g., cTnI and one or more biomarkers other than cTnI (suchas UCH-L1 and/or GFAP)) present in a sample may be as described herein.The methods may also be adapted in view of other methods for analyzinganalytes. Examples of well-known variations include, but are not limitedto, immunoassay, such as sandwich immunoassay (e.g.,monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonalsandwich immunoassays, including enzyme detection (enzyme immunoassay(EIA) or enzyme-linked immunosorbent assay (ELISA), competitiveinhibition immunoassay (e.g., forward and reverse), enzyme multipliedimmunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc.

a. Immunoassay

The analytes of interest, and/or peptides of fragments thereof (e.g.,cTnI and UCH-L1 and/or GFAP, and/or peptides or fragments thereof, i.e.,cTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP) and/or fragments), may be analyzed using antibodies forcTnI and one or more additional biomarkers which are not cTnI (such asUCH-L1 and/or GFAP antibodies) in an immunoassay. The presence or amountof analytes (e.g., cTnI and one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP)) can be determined using antibodies anddetecting specific binding to the analytes (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)). Forexample, the antibody, or antibody fragment thereof, may specificallybind to the analytes (e.g., cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP)). If desired, one or more of theantibodies can be used in combination with one or more commerciallyavailable monoclonal/polyclonal antibodies. Such antibodies areavailable from companies such as R&D Systems, Inc. (Minneapolis, Minn.)and Enzo Life Sciences International, Inc. (Plymouth Meeting, Pa.).

The presence or amount of analytes (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) present in abody sample may be readily determined using an immunoassay, such assandwich immunoassay (e.g., monoclonal-monoclonal sandwich immunoassays,monoclonal-polyclonal sandwich immunoassays, including radioisotopedetection (radioimmunoassay (RIA)) and enzyme detection (enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g.,Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)). An exampleof a point-of-care device that can be used is i-STAT® (Abbott,Laboratories, Abbott Park, Ill.). Other methods that can be used includea chemiluminescent microparticle immunoassay, in particular oneemploying the ARCHITECT® automated analyzer (Abbott Laboratories, AbbottPark, Ill.), as an example. Other methods include, for example, massspectrometry, and immunohistochemistry (e.g., with sections from tissuebiopsies), using anti-analyte (e.g., anti-cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) antibodies(monoclonal, polyclonal, chimeric, humanized, human, etc.) or antibodyfragments thereof against analytes (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)). Other methodsof detection include those described in, for example, U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, each of which is hereby incorporated by reference in itsentirety. Specific immunological binding of the antibody to the analytes(e.g., cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP)) can be detected via direct labels, such asfluorescent or luminescent tags, metals and radionuclides attached tothe antibody or via indirect labels, such as alkaline phosphatase orhorseradish peroxidase.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, the surface of an assay plate (such as microtiter wells),pieces of a solid substrate material, and the like. An assay strip canbe prepared by coating the antibody or plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

A homogeneous format may be used. For example, after the test sample isobtained from a subject, a mixture is prepared. The mixture contains thetest sample being assessed for the analytes (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) and afirst specific binding partner and one or more additional specificbinding partners (such as a second specific binding partner, a thirdspecific binding partner, a fourth specific binding partner, a fifthspecific binding partner, etc.). In some embodiments, the mixturecontains the test sample being assessed for the analytes (e.g., cTnI andone or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP)) and a first specific binding partner, a second specific bindingpartner and a third specific binding partner. The order in which thetest sample, the first specific binding partner, the second specificbinding partner and the third specific binding partner (if present) areadded to form the mixture is not critical. The test sample issimultaneously contacted with the first specific binding partner, thesecond specific binding partner and/or the third specific bindingpartner. In some embodiments, the first specific binding partner and anycTnI contained in the test sample may form a first specific bindingpartner-analyte (e.g., cTnI)-antigen complex and the second specificbinding partner and one or more additional specific binding partners(e.g., the third specific binding partner, the fourth specific bindingpartner, a fifth specific binding partner, etc.) and any earlybiomarkers other than cTnI (e.g., UCH-L1 and/or GFAP) contained in thetest sample may form a second specific binding partner analyte (e.g.,UCH-L1 and/or GFAP)-antigen complex and a third specific binding partneranalyte (e.g., UCH-L1 and/or GFAP)-antigen complex. In some embodiments,the first specific binding partner may bind to cTnI and the secondspecific binding partner may bind to UCH-L1. In some embodiments, thefirst specific binding partner may bind to cTnI and the second specificbinding partner may bind to GFAP. In yet other embodiments, the firstspecific binding partner may bind to cTnI, the second specific bindingpartner may bind to UCH-L1 and the third specific binding partner maybind to GFAP. The first specific binding partner may be an anti-analyteantibody (e.g., anti-cTnI antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous (3) amino acidsof SEQ ID NO: 1 or an anti-UCH-L1 antibody that binds to an epitopehaving an amino acid sequence comprising at least three contiguous aminoacids of SEQ ID NO:2 or an anti-GFAP antibody that binds to an epitopehaving an amino acid sequence comprising at least three contiguous (3)amino acids of SEQ ID NO: 3). The second specific binding partner may bean anti-analyte antibody (e.g., anti-cTnI antibody that binds to anepitope having an amino acid sequence comprising at least threecontiguous (3) amino acids of SEQ ID NO: 1 or an anti-UCH-L1 antibodythat binds to an epitope having an amino acid sequence comprising atleast three contiguous amino acids of SEQ ID NO:2 or an anti-GFAPantibody that binds to an epitope having an amino acid sequencecomprising at least three contiguous (3) amino acids of SEQ ID NO: 3).The third specific binding partner may be an anti-analyte antibody(e.g., anti-cTnI antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 1 or an anti-UCH-L1 antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous amino acids ofSEQ ID NO:2 or an anti-GFAP antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous (3) amino acidsof SEQ ID NO: 3). Moreover, one or more of the first, second and/orthird specific binding partner can be labeled with or contain adetectable label as described above.

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for the analytes (e.g., cTnI andone or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP)) and a first specific binding partner, wherein the first specificbinding partner and any cTnI contained in the test sample form a firstspecific binding partner-analyte (e.g., cTnI)-antigen complex and one ormore additional specific binding partners (such as a second specificbinding partner, a third specific binding partner, a fourth specificbinding partner, a fifth specific binding partner, etc.), wherein theone or more additional specific binding partners and any one or moreearly biomarkers other than cTnI contained in the test sample form a oneor more specific binding partner-analyte (e.g., UCH-L1 and/orGFAP)-antigen complex. In some embodiments, the mixture contains thetest sample being assessed for the analytes (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) and afirst specific binding partner, a second specific binding partner and athird specific binding partner. The order in which the test sample, thefirst specific binding partner, the second specific binding partner andthe third specific binding partner (if present) are added to form themixture is not critical. The test sample is simultaneously contactedwith the first specific binding partner, the second specific bindingpartner and/or the third specific binding partner. In some embodiments,the first specific binding partner and any cTnI contained in the testsample may form a first specific binding partner-analyte (e.g.,cTnI)-antigen complex and the second specific binding partner and one ormore additional specific binding partners (e.g., the third specificbinding partner, the fourth specific binding partner, a fifth specificbinding partner, etc.) and any early biomarkers other than cTnI ((e.g.,UCH-L1 and/or GFAP) contained in the test sample may form a secondspecific binding partner-analyte (e.g., UCH-L1 and/or GFAP)-antigencomplex and/or a third specific binding partner analyte (e.g., UCH-L1and/or GFAP)-antigen complex. In some embodiments, the first specificbinding partner may bind to cTnI and the second specific binding partnermay bind to UCH-L1. In some embodiments, the first specific bindingpartner may bind to cTnI and the second specific binding partner maybind to GFAP. In yet other embodiments, the first specific bindingpartner may bind to cTnI, the second specific binding partner may bindto UCH-L1 and the third specific binding partner may bind to GFAP. Thefirst specific binding partner may be an anti-analyte antibody (e.g.,anti-cTnI antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 1 or an anti-UCH-L1 antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous amino acids ofSEQ ID NO:2 or an anti-GFAP antibody that binds to an epitope having anamino acid sequence comprising at least three contiguous (3) amino acidsof SEQ ID NO: 3). The second specific binding partner may be ananti-analyte antibody (e.g., anti-cTnI antibody that binds to an epitopehaving an amino acid sequence comprising at least three contiguous (3)amino acids of SEQ ID NO: 1 or an anti-UCH-L1 antibody that binds to anepitope having an amino acid sequence comprising at least threecontiguous amino acids of SEQ ID NO:2 or an anti-GFAP antibody thatbinds to an epitope having an amino acid sequence comprising at leastthree contiguous (3) amino acids of SEQ ID NO: 3). The third specificbinding partner may be an anti-analyte antibody (e.g., anti-cTnIantibody that binds to an epitope having an amino acid sequencecomprising at least three contiguous (3) amino acids of SEQ ID NO: 1 oran anti-UCH-L1 antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous amino acids of SEQ ID NO:2or an anti-GFAP antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 3).Moreover, one or more of the first, second and/or third specificbinding partner can be labeled with or contain a detectable label asdescribed above. The order in which the test sample and each of thespecific binding partners (first specific binding partner, secondspecific binding partner, third specific binding partner, fourthspecific binding partner, fifth specific binding partner, etc.) areadded to form the mixture is not critical.

One or more of the specific binding partners (e.g., first specificbinding partner, second specific binding partner, third specific bindingpartner, fourth specific binding partner and/or fifth specific bindingpartner, etc.) may be immobilized on a solid phase. The solid phase usedin the immunoassay (for any of the specific binding partners describedherein) can be any solid phase known in the art, such as, but notlimited to, a magnetic particle, a bead, a test tube, a microtiterplate, a cuvette, a membrane, a scaffolding molecule, a film, a filterpaper, a disc, and a chip. In those embodiments where the solid phase isa bead, the bead may be a magnetic bead or a magnetic particle. Magneticbeads/particles may be ferromagnetic, ferrimagnetic, paramagnetic,superparamagnetic or ferrofluidic. Exemplary ferromagnetic materialsinclude Fe, Co, Ni, Gd, Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examplesof ferrimagnetic materials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (orFeO.Fe₂O₃). Beads can have a solid core portion that is magnetic and issurrounded by one or more non-magnetic layers. Alternately, the magneticportion can be a layer around a non-magnetic core. The solid support onwhich the first specific binding member is immobilized may be stored indry form or in a liquid. The magnetic beads may be subjected to amagnetic field prior to or after contacting with the sample with amagnetic bead on which the first specific binding member is immobilized.

After the mixture containing the one or more specific bindingpartner-analyte (e.g., cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 or GFAP)) antigen complex is formed, any unboundanalytes are removed from the complex using any technique known in theart. For example, the unbound analytes can be removed by washing.Desirably, however, the first specific binding partner is present inexcess of any analytes (e.g., cTnI and UCH-L1 and/or GFAP)) present inthe test sample, such that all analytes (e.g., cTnI and UCH-L1 and/orGFAP)) that are present in the test sample are bound by the firstspecific binding partner.

After any unbound analytes (e.g., cTnI and UCH-L1 and/or GFAP) areremoved, one or more additional specific binding partners (such as asecond specific binding partner, at third specific binding partner, afourth specific binding partner, a fifth specific binding partner, etc.)is added to the mixture to form an additional (namely, second, third,fourth, fifth, etc.) specific binding partner-analyte of interest (e.g.,one or more biomarkers other than cTnI (such as UCH-L 1 and/orGFAP))-additional (namely, second, third, fourth, fifth, etc.) specificbinding partner complex. The additional (namely, second, third, fourth,fifth, etc.) specific binding partner may be an anti-analyte antibody(e.g., an antibody to a biomarker other than cTnI (such as, for example,an anti-UCH-L1 antibody that binds to an epitope having an amino acidsequence comprising at least three contiguous (3) amino acids of SEQ IDNO: 2 and/or anti-GFAP antibody that binds to an epitope having an aminoacid sequence comprising at least three contiguous (3) amino acids ofSEQ ID NO: 3). Moreover, the additional (namely, second, third, fourth,fifth, etc.) specific binding partner is labeled with or contains adetectable label as described above.

The use of immobilized antibodies or antibody fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles (such as a magnetic bead), latex particles or modified surfacelatex particles, polymer or polymer film, plastic or plastic film,planar substrate, the surface of an assay plate (such as microtiterwells), pieces of a solid substrate material, and the like. An assaystrip can be prepared by coating the antibody or plurality of antibodiesin an array on a solid support. This strip can then be dipped into thetest sample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

(1) Sandwich Immunoassay

A sandwich immunoassay measures the amount of antigen between two layersof antibodies (i.e., at least one capture antibody) and a detectionantibody (e.g., at least one detection antibody). For each analyte ofinterest, such as cTnI or one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP, to be detected, the capture antibody andthe detection antibody bind to different epitopes on the antigen, e.g.,analyte of interest such as cTnI or one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP). Desirably, binding of thecapture antibody to an epitope does not interfere with binding of thedetection antibody to an epitope. Either monoclonal or polyclonalantibodies may be used as the capture and detection antibodies in thesandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte (e.g., cTnI and one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP)) in a test sample. More specifically, theat least two antibodies bind to certain epitopes of each analyte (e.g.,cTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP)) forming an immune complex which is referred to as a“sandwich”. One or more antibodies can be used to capture the analyte(e.g., cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP)) in the test sample (these antibodies are frequentlyreferred to as a “capture” antibody or “capture” antibodies) and one ormore antibodies is used to bind a detectable (namely, quantifiable)label to the sandwich (these antibodies are frequently referred to asthe “detection” antibody or “detection” antibodies). In a sandwichassay, the binding of an antibody to its epitope desirably is notdiminished by the binding of any other antibody in the assay to itsrespective epitope. Antibodies are selected so that the one or morefirst antibodies brought into contact with a test sample suspected ofcontaining analytes (e.g., cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP)) do not bind to all or part of anepitope recognized by the second or subsequent antibodies, therebyinterfering with the ability of the one or more second detectionantibodies to bind to the analytes (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)).

The antibodies may be used as a first antibody in said immunoassay. Theantibody immunospecifically binds to epitopes on the analyte (e.g., cTnIor one or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP)). In addition to the antibodies of the present disclosure, saidimmunoassay may comprise a second antibody that immunospecifically bindsto epitopes that are not recognized or bound by the first antibody.

A test sample suspected of containing analytes (e.g., cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) canbe contacted with at least one first capture antibody (or antibodies)and at least one second detection antibody (or antibodies) for eachanalyte either simultaneously or sequentially. In the sandwich assayformat, a test sample suspected of containing analytes (e.g., cTnI andone or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP)) is first brought into contact with the at least one first captureantibody for each analyte that specifically binds to a particularepitope under conditions which allow the formation of a firstantibody-analyte (e.g., cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP)) antigen complex. If more than onecapture antibody is used, a first multiple capture antibody-antigen(such as cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP)) complex is formed. In a sandwich assay, theantibodies, preferably, the at least one capture antibody for eachanalyte, are used in molar excess amounts of the maximum amount of eachanalyte (e.g., cTnI and one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP)) expected in the test sample. For example,from about 5 μg/mL to about 1 mg/mL of antibody per ml of microparticlecoating buffer may be used.

i. Anti-cTnI Capture Antibody

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one first capture antibody can bebound to a solid support which facilitates the separation the firstantibody-analyte (e.g., cTnI) complex from the test sample. Any solidsupport known in the art can be used, including but not limited to,solid supports made out of polymeric materials in the forms of wells,tubes, or beads (such as a microparticle). The antibody (or antibodies)can be bound to the solid support by adsorption, by covalent bondingusing a chemical coupling agent or by other means known in the art,provided that such binding does not interfere with the ability of theantibody to bind analyte (e.g., cTnI). Moreover, if necessary, the solidsupport can be derivatized to allow reactivity with various functionalgroups on the antibody. Such derivatization requires the use of certaincoupling agents such as, but not limited to, maleic anhydride,N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte (e.g., cTnI) isincubated in order to allow for the formation of a first captureantibody (or multiple antibody)-analyte (e.g., cTnI) complex. Theincubation can be carried out at a pH of from about 4.5 to about 10.0,at a temperature of from about 2° C. to about 45° C., and for a periodfrom at least about one (1) minute to about eighteen (18) hours, fromabout 2-6 minutes, from about 7-12 minutes, from about 5-15 minutes, orfrom about 3-4 minutes.

ii. Anti-UCH-L1 and/or GFAP Capture Antibodies

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one first capture antibody can bebound to a solid support which facilitates the separation the firstantibody-analyte (e.g., UCH-L1 and/or GFAP) complex from the testsample. Any solid support known in the art can be used, including butnot limited to, solid supports made out of polymeric materials in theforms of wells, tubes, or beads (such as a microparticle). The antibody(or antibodies) can be bound to the solid support by adsorption, bycovalent bonding using a chemical coupling agent or by other means knownin the art, provided that such binding does not interfere with theability of the antibody to bind analyte (e.g., UCH-L1 and/or GFAP).Moreover, if necessary, the solid support can be derivatized to allowreactivity with various functional groups on the antibody. Suchderivatization requires the use of certain coupling agents such as, butnot limited to, maleic anhydride, N-hydroxysuccinimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte (e.g., UCH-L1and/or GFAP) is incubated in order to allow for the formation of a firstcapture antibody (or multiple antibody)-analyte (e.g., UCH-L1 and/orGFAP) complex. The incubation can be carried out at a pH of from about4.5 to about 10.0, at a temperature of from about 2° C. to about 45° C.,and for a period from at least about one (1) minute to about eighteen(18) hours, from about 2-6 minutes, from about 7-12 minutes, from about5-15 minutes, or from about 3-4 minutes.

iii. Detection Antibody

After formation of the first/multiple capture antibody-analyte (e.g.,cTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP)) complex, the complex is then contacted with at least onesecond detection antibody (under conditions that allow for the formationof a first/multiple antibody-analyte (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) antigen-secondantibody complex). In some embodiments, the test sample is contactedwith the detection antibody simultaneously with the capture antibody. Ifthe first antibody-analyte (e.g., cTnI and one or more early biomarkersother than cTnI (such as UCH-L1 and/or GFAP)) complex is contacted withmore than one detection antibody, then a first/multiple captureantibody-analyte (e.g., cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP))-multiple antibody detection complexis formed. As with first antibody, when the at least second (andsubsequent) antibody is brought into contact with the firstantibody-analyte (e.g., cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP)) complex, a period of incubation underconditions similar to those described above is required for theformation of the first/multiple antibody-analyte (e.g., cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/orGFAP))-second/multiple antibody complex. Preferably, at least one secondantibody contains a detectable label. The detectable label can be boundto the at least one second antibody prior to, simultaneously with orafter the formation of the first/multiple antibody-analyte (e.g., cTnIand one or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP))-second/multiple antibody complex. Any detectable label known inthe art can be used.

Chemiluminescent assays can be performed in accordance with the methodsdescribed in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006).While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly. The chemiluminometer can be equipped with multiple reagentinjectors using 96-well black polystyrene microplates (Costar #3792).Each sample can be added into a separate well, followed by thesimultaneous/sequential addition of other reagents as determined by thetype of assay employed. Desirably, the formation of pseudobases inneutral or basic solutions employing an acridinium aryl ester isavoided, such as by acidification. The chemiluminescent response is thenrecorded well-by-well. In this regard, the time for recording thechemiluminescent response will depend, in part, on the delay between theaddition of the reagents and the particular acridinium employed.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith an acridinium compound, detectably labeled first specific bindingpartner-antigen (e.g., cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP)) complexes form. Alternatively, if asecond specific binding partner is used and the second specific bindingpartner is detectably labeled with an acridinium compound, detectablylabeled first specific binding partner-analyte (e.g., cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/orGFAP))-second specific binding partner complexes form. Any unboundspecific binding partner, whether labeled or unlabeled, can be removedfrom the mixture using any technique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture before, simultaneously with, or after theaddition of an above-described acridinium compound. Hydrogen peroxidecan be generated in situ in a number of ways such as would be apparentto one skilled in the art.

Alternatively, a source of hydrogen peroxide can be simply added to themixture. For example, the source of the hydrogen peroxide can be one ormore buffers or other solutions that are known to contain hydrogenperoxide. In this regard, a solution of hydrogen peroxide can simply beadded.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte (e.g., cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) isgenerated. The basic solution contains at least one base and has a pHgreater than or equal to 10, preferably, greater than or equal to 12.Examples of basic solutions include, but are not limited to, sodiumhydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide,magnesium hydroxide, sodium carbonate, sodium bicarbonate, calciumhydroxide, calcium carbonate, and calcium bicarbonate. The amount ofbasic solution added to the sample depends on the concentration of thebasic solution. Based on the concentration of the basic solution used,one skilled in the art can easily determine the amount of basic solutionto add to the sample. Other labels other than chemiluminescent labelscan be employed. For instance, enzymatic labels (including but notlimited to alkaline phosphatase) can be employed.

The chemiluminescent signal, or other signal, that is generated can bedetected using routine techniques known to those skilled in the art.Based on the intensity of the signal generated, the amount of theanalytes of interest (e.g., cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP)) in the sample can be quantified.Specifically, the amount of the analytes (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) in thesample is proportional to the intensity of the signal generated. Theamount of analytes (e.g., cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP)) present can be quantified bycomparing the amount of light generated to a standard curve for analyte(e.g., cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP)) or by comparison to a reference standard. Thestandard curve can be generated using serial dilutions or solutions ofknown concentrations of the analyte (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) by massspectroscopy, gravimetric methods, and other techniques known in theart.

(2) Forward Competitive Inhibition Assay

In a forward competitive format, an aliquot of labeled the analytes ofinterest (e.g., cTnI and one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP)) having a fluorescent label, a tag attachedwith a cleavable linker, etc.) of a known concentration is used tocompete with the analytes of interest (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) in a testsample for binding to the analytes of interest antibody (e.g., a cTnIantibody and one or more additional antibodies which do not bind to acTnI (such as an UCH-L1 and/or GFAP antibody)).

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and labeled analytes of interest,analytes of interest fragment or analytes of interest variant thereof.The analytes of interest peptide, analytes of interest fragment oranalytes of interest variant can be labeled with any detectable label,including a detectable label comprised of tag attached with a cleavablelinker. In this assay, the antibody for each analyte can be immobilizedon to a solid support. Alternatively, the antibody for each analyte canbe coupled to an antibody, such as an antispecies antibody, that hasbeen immobilized on a solid support, such as a microparticle or planarsubstrate.

The labeled analytes of interest, the test sample and the antibody foreach analyte are incubated under conditions similar to those describedabove in connection with the sandwich assay format. Two differentspecies of antibody-analyte of interest complexes may then be generatedfor each analyte. Specifically, one of the antibody-analyte of interestcomplexes generated contains a detectable label (e.g., a fluorescentlabel, etc.) while the other antibody-analyte of interest complex doesnot contain a detectable label. The antibody-analyte of interest complexcan be, but does not have to be, separated from the remainder of thetest sample prior to quantification of the detectable label. Regardlessof whether the antibody-analyte of interest complex is separated fromthe remainder of the test sample, the amount of detectable label in theantibody-analyte of interest complex is then quantified. Theconcentration of the analytes of interest (such as membrane-associatedanalytes of interest, soluble analytes of interest, fragments of solubleanalytes of interest, variants of analytes of interest(membrane-associated or soluble analytes of interest) or anycombinations thereof) in the test sample can then be determined, e.g.,as described above.

(3) Reverse Competitive Inhibition Assay

In a reverse competition assay, an immobilized analytes of interest(e.g., cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP)) can either be sequentially or simultaneouslycontacted with a test sample and at least one labeled antibody.

The analytes of interest can be bound to a solid support, such as thesolid supports discussed above in connection with the sandwich assayformat.

The immobilized analytes of interest, test sample and at least onelabeled antibody for each analyte are incubated under conditions similarto those described above in connection with the sandwich assay format.Two different species analytes of interest-antibody complexes are thengenerated for each analyte. Specifically, for each analyte one of theanalyte of interest-antibody complexes generated is immobilized andcontains a detectable label (e.g., a fluorescent label, etc.) while theother analyte of interest-antibody complex is not immobilized andcontains a detectable label. The non-immobilized analyte ofinterest-antibody complex and the remainder of the test sample areremoved from the presence of the immobilized analyte ofinterest-antibody complex through techniques known in the art, such aswashing. Once the non-immobilized analyte of interest antibody complexis removed, the amount of detectable label in the immobilized analyte ofinterest-antibody complex is then quantified following cleavage of thetag. The concentration of each analyte of interest in the test samplecan then be determined by comparing the quantity of detectable label asdescribed above.

(4) One-Step Immunoassay or “Capture on the Fly” Assay

In a capture on the fly immunoassay, a solid substrate is pre-coatedwith an immobilization agent. The capture agent for each analyte (e.g.,cTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP)) and the detection agent for each analyte are added to thesolid substrate together, followed by a wash step prior to detection.The capture agent can bind the analyte (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)) and comprises aligand for an immobilization agent. The capture agent and the detectionagents may be antibodies or any other moiety capable of capture ordetection as described herein or known in the art. The ligand maycomprise a peptide tag and an immobilization agent may comprise ananti-peptide tag antibody. Alternately, the ligand and theimmobilization agent may be any pair of agents capable of bindingtogether so as to be employed for a capture on the fly assay (e.g.,specific binding pair, and others such as are known in the art). Morethan one analyte may be measured. In some embodiments, the solidsubstrate may be coated with an antigen and the analyte to be analyzedis an antibody.

In certain other embodiments, in a one-step immunoassay or “capture onthe fly”, a solid support (such as a microparticle) pre-coated with animmobilization agent (such as biotin, streptavidin, etc.) and at least afirst specific binding member and a second specific binding member(which function as capture and detection reagents, respectively) areused. The first specific binding member comprises a ligand for theimmobilization agent (for example, if the immobilization agent on thesolid support is streptavidin, the ligand on the first specific bindingmember may be biotin) and also binds to the analyte of interest (e.g.,cTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP)). The second specific binding member comprises a detectablelabel and binds to an analyte of interest (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)). Thesolid support and the first and second specific binding members may beadded to a test sample (either sequentially or simultaneously). Theligand on the first specific binding member binds to the immobilizationagent on the solid support to form a solid support/first specificbinding member complex. Any analyte of interest present in the samplebinds to the solid support/first specific binding member complex to forma solid support/first specific binding member/analyte complex. Thesecond specific binding member binds to the solid support/first specificbinding member/analyte complex and the detectable label is detected. Anoptional wash step may be employed before the detection. In certainembodiments, in a one-step assay more than one analyte may be measured.In certain other embodiments, more than two specific binding members canbe employed. In certain other embodiments, multiple detectable labelscan be added. In certain other embodiments, multiple analytes ofinterest can be detected, or their amounts, levels or concentrations,measured, determined or assessed.

The use of a capture on the fly assay can be done in a variety offormats as described herein, and known in the art. For example, theformat can be a sandwich assay such as described above, but alternatelycan be a competition assay, can employ a single specific binding member,or use other variations such as are known.

13. OTHER FACTORS

The methods of diagnosing, prognosticating, and/or assessing, asdescribed above, can further include using other factors for thediagnosis, prognostication, and assessment. In some embodiments,traumatic brain injury may be diagnosed using the Glasgow Coma Scale orthe Extended Glasgow Outcome Scale (GOSE). Other tests, scales orindices can also be used either alone or in combination with the GlasgowComa Scale. An example is the Ranchos Los Amigos Scale. The Ranchos LosAmigos Scale measures the levels of awareness, cognition, behavior andinteraction with the environment. The Ranchos Los Amigos Scale includes:Level I: No Response; Level II: Generalized Response; Level III:Localized Response; Level IV: Confused-agitated; Level V:Confused-inappropriate; Level VI: Confused-appropriate; Level VII:Automatic-appropriate; and Level VIII: Purposeful-appropriate.

14. SAMPLES

In some embodiments, the sample is obtained after the human subjectsustained an injury to the head caused by physical shaking, blunt impactby an external mechanical or other force that results in a closed oropen head trauma, one or more falls, explosions or blasts or other typesof blunt force trauma. In some embodiments, the sample is obtained afterthe human subject has ingested or been exposed to a chemical, toxin orcombination of a chemical and toxin. Examples of such chemicals and/ortoxins include, fires, molds, asbestos, pesticides and insecticides,organic solvents, paints, glues, gases (such as carbon monoxide,hydrogen sulfide, and cyanide), organic metals (such as methyl mercury,tetraethyl lead and organic tin) and/or one or more drugs of abuse. Insome embodiments, the sample is obtained from a human subject thatsuffers from an autoimmune disease, a metabolic disorder, a brain tumor,hypoxia, one or more viruses, meningitis, hydrocephalus or combinationsthereof.

In yet another embodiment, the methods described herein use samples thatalso can be used to determine whether or not a subject has or is at riskof developing mild traumatic brain injury by determining the levels ofcTnI and one or more early biomarkers other than cTnI (such as UCH-L1and/or GFAP) in a subject using the anti-cTnI antibodies (and theanti-UCH-L1 and/or anti-GFAP antibodies) described below, or antibodyfragments thereof. Thus, in particular embodiments, the disclosure alsoprovides a method for determining whether a subject having, or at riskfor, traumatic brain injuries, discussed herein and known in the art, isa candidate for therapy or treatment. Generally, the subject is at leastone who: (i) has experienced an injury to the head; (ii) ingested and/orbeen exposed to one or more chemicals and/or toxins; (iii) suffers froman autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, oneor more viruses, meningitis, hydrocephalus or suffers from anycombinations thereof; or (iv) any combinations of (i)-(iii); or, who hasactually been diagnosed as having, or being at risk for TBI (such as,for example, subjects suffering from an autoimmune disease, a metabolicdisorder, a brain tumor, hypoxia, one or more viruses, meningitis,hydrocephalus or combinations thereof), and/or who demonstrates anunfavorable (i.e., clinically undesirable) concentration or amount ofcTnI or cTnI fragment and one or more early biomarkers other than cTnI(such as UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragment), asdescribed herein.

a. Test or Biological Sample

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP). Thesample may be derived from any suitable source. In some cases, thesample may comprise a liquid, fluent particulate solid, or fluidsuspension of solid particles. In some cases, the sample may beprocessed prior to the analysis described herein. For example, thesample may be separated or purified from its source prior to analysis;however, in certain embodiments, an unprocessed sample containing cTnIand one or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP) may be assayed directly. In a particular example, the sourcecontaining cTnI and one or more early biomarkers other than cTnI (suchas UCH-L1 and/or GFAP) is a human bodily substance (e.g., bodily fluid,blood such as whole blood, serum, plasma, urine, saliva, sweat, sputum,semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitialfluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or thelike). Tissues may include, but are not limited to skeletal muscletissue, liver tissue, lung tissue, kidney tissue, myocardial tissue,brain tissue, bone marrow, cervix tissue, skin, etc. The sample may be aliquid sample or a liquid extract of a solid sample. In certain cases,the source of the sample may be an organ or tissue, such as a biopsysample, which may be solubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or thelike. In some cases, the volume of the fluid sample is between about0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, betweenabout 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source containing cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP) is ahuman body fluid (e.g., whole blood, serum or plasma), the fluid may bediluted with an appropriate solvent (e.g., a buffer such as PBS buffer).A fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold,or greater, prior to use. In other cases, the fluid sample is notdiluted prior to use in an assay.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source containing cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) is a human bodyfluid (e.g., blood, serum), the fluid may be concentrated byprecipitation, evaporation, filtration, centrifugation, or a combinationthereof. A fluid sample may be concentrated about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold,about 100-fold, or greater, prior to use.

b. Controls

It may be desirable to include a control sample. The control sample maybe analyzed concurrently with the sample from the subject as describedabove. The results obtained from the subject sample can be compared tothe results obtained from the control sample. Standard curves may beprovided, with which assay results for the sample may be compared. Suchstandard curves present levels of marker as a function of assay units,i.e., fluorescent signal intensity, if a fluorescent label is used.Using samples taken from multiple donors, standard curves can beprovided for reference levels of the cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) in normalhealthy tissue, as well as for “at-risk” levels of the cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP) intissue taken from donors, who may have one or more of thecharacteristics set forth above.

Thus, in view of the above, a method for determining the presence,amount, or concentration of cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP) in a test sample is provided. Themethod comprises assaying the test sample for cTnI and UCH-L1 and/orGFAP by an immunoassay, for example, employing at least one captureantibody that binds to an epitope on cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) and at least onedetection antibody that binds to an epitope on cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP) which isdifferent from the epitope for the capture antibody and optionallyincludes a detectable label, and comprising comparing a signal generatedby the detectable label as a direct or indirect indication of thepresence, amount or concentration of cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) in the testsample to a signal generated as a direct or indirect indication of thepresence, amount or concentration of cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) in a calibrator.The calibrator is optionally, and is preferably, part of a series ofcalibrators in which each of the calibrators differs from the othercalibrators in the series by the concentration of cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP).

15. KIT

Provided herein is a kit, which may be used for assaying or assessing atest sample for cTnI and/or cTnI fragment and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP or UCH-L1 and/orGFAP fragment). The kit comprises at least one component for assayingthe test sample for cTnI and instructions for assaying the test samplecontaining cTnI and instructions for assaying the test sample for one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP). Forexample, the kit can comprise instructions for assaying the test samplefor cTnI and one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP) by immunoassay, e.g., chemiluminescent microparticleimmunoassay. Instructions included in kits can be affixed to packagingmaterial or can be included as a package insert. While the instructionsare typically written or printed materials they are not limited to such.Any medium capable of storing such instructions and communicating themto an end user is contemplated by this disclosure. Such media include,but are not limited to, electronic storage media (e.g., magnetic discs,tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.As used herein, the term “instructions” can include the address of aninternet site that provides the instructions.

The at least one component may include at least one compositioncomprising one or more isolated antibodies or antibody fragments thereofthat specifically bind to cTnI and one or more early biomarkers otherthan cTnI (such as UCH-L1 and/or GFAP). The antibody may be a cTnIdetection antibody and/or capture antibody and/or an antibody that bindsto one or more biomarkers other than cTnI such as an UCH-L1 and/or GFAPcapture antibody and/or a UCH-L1 and/or GFAP detection antibody).

Alternatively or additionally, the kit can comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized, cTnI and one ormore early biomarkers other than cTnI (such as UCH-L1 and/or GFAP)and/or at least one container (e.g., tube, microtiter plates or strips,which can be already coated with an anti-cTnI antibody or an antibodyother than an anti-cTnI antibody (such as an anti-UCH-L1 and/or GFAPmonoclonal antibody)) for conducting the assay, and/or a buffer, such asan assay buffer or a wash buffer, either one of which can be provided asa concentrated solution, a substrate solution for the detectable label(e.g., an enzymatic label), or a stop solution. Preferably, the kitcomprises all components, i.e., reagents, standards, buffers, diluents,etc., which are necessary to perform the assay. The instructions alsocan include instructions for generating a standard curve.

The kit may further comprise reference standards for quantifying cTnIand one or more early biomarkers other than cTnI (such as UCH-L1 and/orGFAP). The reference standards may be employed to establish standardcurves for interpolation and/or extrapolation of cTnI concentrations andconcentrations of one or more early biomarkers other than cTnI (such asUCH-L1 and/or GFAP). In some embodiments, the reference standards forcTnI can correspond to the 99th percentile derived from a healthyreference population. Such reference standards can be determined usingroutine techniques known in the art. The reference standards may includea high UCH-L1 and/or GFAP concentration level, for example, about 100000pg/mL, about 125000 pg/mL, about 150000 pg/mL, about 175000 pg/mL, about200000 pg/mL, about 225000 pg/mL, about 250000 pg/mL, about 275000pg/mL, or about 300000 pg/mL; a medium UCH-L1 and/or GFAP concentrationlevel, for example, about 25000 pg/mL, about 40000 pg/mL, about 45000pg/mL, about 50000 pg/mL, about 55000 pg/mL, about 60000 pg/mL, about75000 pg/mL or about 100000 pg/mL; and/or a low UCH-L1 and/or GFAPconcentration level, for example, about 1 pg/mL, about 5 pg/mL, about 10pg/mL, about 12.5 pg/mL, about 15 pg/mL, about 20 pg/mL, about 25 pg/mL,about 30 pg/mL, about 35 pg/mL, about 40 pg/mL, about 45 pg/mL, about 50pg/mL, about 55 pg/mL, about 60 pg/mL, about 65 pg/mL, about 70 pg/mL,about 75 pg/mL, about 80 pg/mL, about 85 pg/mL, about 90 pg/mL, about 95pg/mL, or about 100 pg/mL.

Any antibodies, which are provided in the kit, such as recombinantantibodies specific for cTnI and/or for one or more antibodies specificor one or more biomarkers other than cTnI (such as UCH-L1 and/or GFAP),can incorporate a detectable label, such as a fluorophore, radioactivemoiety, enzyme, biotin/avidin label, chromophore, chemiluminescentlabel, or the like, or the kit can include reagents for labeling theantibodies or reagents for detecting the antibodies (e.g., detectionantibodies) and/or for labeling the analytes (e.g., cTnI and one or moreearly biomarkers other than cTnI (such as UCH-L1 and/or GFAP)) orreagents for detecting the analyte (e.g., cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP)). Theantibodies, calibrators, and/or controls can be provided in separatecontainers or pre-dispensed into an appropriate assay format, forexample, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays,

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine, whole blood, plasma, or serum sample). Whereappropriate, the kit optionally also can contain reaction vessels,mixing vessels, and other components that facilitate the preparation ofreagents or the test sample. The kit can also include one or moreinstrument for assisting with obtaining a test sample, such as asyringe, pipette, forceps, measured spoon, or the like.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper,disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of traumatic brain injury or disorder.

a. Adaptation of Kit and Method

The kit (or components thereof), as well as the method for assessing ordetermining the concentration of cTnI and one or more early biomarkersother than cTnI (such as UCH-L1 and/or GFAP) in a test sample by animmunoassay as described herein, can be adapted for use in a variety ofautomated and semi-automated systems (including those wherein the solidphase comprises a microparticle), as described, e.g., U.S. Pat. No.5,063,081, U.S. Patent Application Publication Nos. 2003/0170881,2004/0018577, 2005/0054078, and 2006/0160164 and as commerciallymarketed e.g., by Abbott Laboratories (Abbott Park, Ill.) as AbbottPoint of Care (i-STAT® or i-STAT Alinity, Abbott Laboratories) as wellas those described in U.S. Pat. Nos. 5,089,424 and 5,006,309, and ascommercially marketed, e.g., by Abbott Laboratories (Abbott Park, Ill.)as ARCHITECT® or the series of Abbott Alinity devices.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., analyte antibody orcapture antibody) is attached (which can affect sandwich formation andanalyte reactivity), and the length and timing of the capture,detection, and/or any optional wash steps. Whereas a non-automatedformat such as an ELISA may require a relatively longer incubation timewith sample and capture reagent (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT® and any successor platform,Abbott Laboratories) may have a relatively shorter incubation time(e.g., approximately 18 minutes for ARCHITECT®). Similarly, whereas anon-automated format such as an ELISA may incubate a detection antibodysuch as the conjugate reagent for a relatively longer incubation time(e.g., about 2 hours), an automated or semi-automated format (e.g.,ARCHITECT® and any successor platform) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT® andany successor platform).

Other platforms available from Abbott Laboratories include, but are notlimited to, immunoassay analyzers including AxSYM®, IMx® (see, e.g.,U.S. Pat. No. 5,294,404, which is hereby incorporated by reference inits entirety), PRISM®, EIA (bead), and Quantum™ II (dual-wavelengthanalyzer system), as well as other platforms. Additionally, the assays,kits, and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems. Asmentioned previously, the present disclosure is, for example, applicableto the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories)electrochemical immunoassay system that performs sandwich immunoassays.Immunosensors and their methods of manufacture and operation insingle-use test devices are described, for example in, U.S. Pat. No.5,063,081, U.S. Patent App. Publication Nos. 2003/0170881, 2004/0018577,2005/0054078, and 2006/0160164, which are incorporated in theirentireties by reference for their teachings regarding same.

In particular, with regard to the adaptation of an assay to the i-STAT®system, the following configuration is preferred. A microfabricatedsilicon chip is manufactured with a pair of gold amperometric workingelectrodes and a silver-silver chloride reference electrode. On one ofthe working electrodes, polystyrene beads (0.2 mm diameter) withimmobilized capture antibody are adhered to a polymer coating ofpatterned polyvinyl alcohol over the electrode. This chip is assembledinto an i-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the silicon chip, there is a specificbinding partner for cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP), such as one or more cTnI antibodiesone or more monoclonal/polyclonal antibody or a fragment thereof, avariant thereof, or a fragment of a variant thereof that can bind cTnI)or one or more anti-cTnI DVD-Igs (or a fragment thereof, a variantthereof, or a fragment of a variant thereof that can bind cTnI) and oneor more antibodies specific for one or more biomarkers other than cTnI(such as UCH-L1 and/or GFAP antibodies (one or moremonoclonal/polyclonal antibody or a fragment thereof, a variant thereof,or a fragment of a variant thereof that can bind UCH-L1 and/or GFAP) orone or more anti-UCH-L1 and/or anti-GFAP DVD-Igs (or a fragment thereof,a variant thereof, or a fragment of a variant thereof that can bindUCH-L1 and/or GFAP and/or GFAP), any of which can be detectably labeled.Within the fluid pouch of the cartridge is an aqueous reagent thatincludes p-aminophenol phosphate.

In operation, a sample from a subject suspected of suffering from TBI isadded to the holding chamber of the test cartridge, and the cartridge isinserted into the i-STAT® reader. A pump element within the cartridgepushes the sample into a conduit containing the chip. The sample isbrought into contact with the sensors allowing the enzyme conjugate todissolve into the sample. The sample is oscillated across the sensors topromote formation of the sandwich of approximately 2-12 minutes. In thepenultimate step of the assay, the sample is pushed into a waste chamberand wash fluid, containing a substrate for the alkaline phosphataseenzyme, is used to wash excess enzyme conjugate and sample off thesensor chip. In the final step of the assay, the alkaline phosphataselabel reacts with p-aminophenol phosphate to cleave the phosphate groupand permit the liberated p-aminophenol to be electrochemically oxidizedat the working electrode. Based on the measured current, the reader isable to calculate the amount of cTnI and one or more early biomarkersother than cTnI (such as UCH-L1 and/or GFAP) in the sample by means ofan embedded algorithm and factory-determined calibration curve.

The methods and kits as described herein necessarily encompass otherreagents and methods for carrying out the immunoassay. For instance,encompassed are various buffers such as are known in the art and/orwhich can be readily prepared or optimized to be employed, e.g., forwashing, as a conjugate diluent, and/or as a calibrator diluent. Anexemplary conjugate diluent is ARCHITECT® conjugate diluent employed incertain kits (Abbott Laboratories, Abbott Park, Ill.) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.), which comprises a buffer containingMES, other salt, a protein blocker, and an antimicrobial agent.Additionally, as described in U.S. Patent Application No. 61/142,048filed Dec. 31, 2008, improved signal generation may be obtained, e.g.,in an i-STAT® cartridge format, using a nucleic acid sequence linked tothe signal antibody as a signal amplifier.

While certain embodiments herein are advantageous when employed toassess disease, such as traumatic brain injury, the assays and kits alsooptionally can be employed to assess cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) in otherdiseases, disorders, and conditions as appropriate.

The method of assay also can be used to identify a compound thatameliorates diseases, such as traumatic brain injury. For example, acell that expresses cTnI and one or more early biomarkers other thancTnI (such as UCH-L1 and/or GFAP) can be contacted with a candidatecompound. The level of expression of cTnI and one or more earlybiomarkers other than cTnI (such as UCH-L1 and/or GFAP) in the cellcontacted with the compound can be compared to that in a control cellusing the method of assay described herein.

The present disclosure has multiple aspects, illustrated by thefollowing non-limiting examples.

16. EXAMPLES

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the presentdisclosure described herein are readily applicable and appreciable, andmay be made using suitable equivalents without departing from the scopeof the present disclosure or the aspects and embodiments disclosedherein. Having now described the present disclosure in detail, the samewill be more clearly understood by reference to the following examples,which are merely intended only to illustrate some aspects andembodiments of the disclosure, and should not be viewed as limiting tothe scope of the disclosure. The disclosures of all journal references,U.S. patents, and publications referred to herein are herebyincorporated by reference in their entireties.

The present disclosure has multiple aspects, illustrated by thefollowing non-limiting examples.

Example 1 i-STAT® UCH-L1 Assay

Monoclonal antibody pairs, such as Antibody A as a capture monoclonalantibody and Antibody B and C as a detection monoclonal antibody, weretested. Antibody A is an exemplary anti-UCH-L1 antibody that wasinternally developed at Abbott Laboratories (Abbott Park, Ill.).Antibody B and C recognize different epitopes of UCH-L1 and enhance thedetection of antigen in the sample that were developed by BanyanBiomarkers (Alachua, Fla.). Other antibodies that were internallydeveloped at Abbott Laboratories (Abbott Park, Ill.) also show or areexpected to show similar enhancement of signal when used together ascapture antibodies or detection antibodies, in various combinations. TheUCH-L1 assay design was evaluated against key performance attributes.The cartridge configuration was Antibody Configuration: Antibody A(Capture Antibody)/Antibody B+C (Detection Antibody); Reagentconditions: 0.8% solids, 125 μg/mL Fab Alkaline Phosphatase clusterconjugate; and Sample Inlet Print: UCH-L1 standard. The assay time was10-15 min (with 7-12 min sample capture time). The i-STAT UCH-L1 assaywas used in a TBI patient population study.

Example 2 i-STAT® GFAP Assay

The i-STAT® GFAP assay was used in a TBI patient population study.Monoclonal antibody pairs, such as Antibody A as a capture monoclonalantibody and Antibody B as a detection monoclonal antibody, were used.Antibody A and Antibody B are exemplary anti-GFAP antibodies that wereinternally developed at Abbott Laboratories (Abbott Park, Ill.). TheGFAP assay design was evaluated against key performance attributes. Thecartridge configuration was Antibody Configuration: Antibody A (CaptureAntibody)/Antibody B (Detection Antibody); Reagent conditions: 0.8%solids, 250 μg/mL Fab Alkaline Phosphatase cluster conjugate; and SampleInlet Print: GFAP specific. The assay time was 10-15 min (with 7-12 minsample capture time).

Example 3 Study 1—TBI Population

Study 1 was a large and complex project. Its institutional andpublic-private partnership is comprised of over 11 clinical sites, 7Cores, for a total of nearly 50 collaborating institutions,corporations, and philanthropy. An earlier Pilot study, based onclinical data from three clinical sites, helped refine TBI Common DataElements and created a prototype of the TBI Information Commons forStudy 1.

Subject Groups: A total of 2,700 to 3000 TBI patients were enrolledevenly across 3 clinical groups, differentiated by clinical carepath: 1. Patients evaluated in the Emergency Department and discharged(ED); 2. Patients admitted to the hospital, but not to ICU (ADM); and 3.Patients admitted to the ICU (ICU). An additional 100 patients perclinical group (n=300) with extracranial trauma but no TBI were enrolledas controls for a total enrollment of 3000 patients. This stratificationplan facilitated comparative effectiveness research (CER) analysis andwas not constrained by traditional differentiation into“Mild/Moderate/Severe” TBI. Data collection was dependent on theclinical care path (ED, ADM, ICU) and requirements of each aim. Patientsin each group were stratified into 3 cohorts that define the extent ofdata to be collected.

The controls were adult orthopedic trauma patients who met the followingcriteria: 1. An Abbreviated Injury Score of ≤4 (not life threatening)for their extremity and/or pelvis injury and/or rib fracture; 2. Met thesame inclusion and exclusion criteria as the TBI subjects except thatthe criterion of having undergone a CT or MRI in the ED for suspectedhead injury did not apply. TBI was ruled out for the current injury byinterviewing potential controls about loss of consciousness (LOC),disturbance of consciousness, and posttraumatic amnesia (PTA)/RA; 3.Each site was provided a plan for the number of controls to targetaccording to age and gender distributions derived from the TBI Cohort;and 4. Controls were enrolled into the CA-MRI cohort for follow-up anddrop to comprehensive assessment (CA) at 2-weeks if unable to completethe MRI visit.

Subject Eligibility: Adult patients were enrolled of all ages presentingto the Emergency Department (ED) with a history of acute TBI as perAmerican Congress of Rehabilitation Medicine (ACRM) Criteria, in whichthe patient had sustained a traumatically induced physiologicaldisruption of brain function, as manifested by ≥one of the following:any period of loss of consciousness (LOC); any loss of memory for events(e.g., amnesia) immediately before or after the accident; any alterationof mental state at the time of the accident (feeling dazed, disoriented,and/or confused); and/or focal neurologic deficits that may or may notbe permanent. Traumatically induced included the head being struck, thehead striking an object, or the brain undergoing anacceleration/deceleration movement (e.g., whiplash) without directexternal trauma to the head.

The Inclusion/Exclusion Criteria used is shown in Table 2.

TABLE 2 Data Criterion Source Comments Inclusion Criteria 1. Age 0-100Chart 2. Documented/verified TBI Chart,  (ACRM Criteria) Inter- view 3.Injury occurred < 24 hours Chart,  ago Inter- view 4. Acute brain CT forclinical Chart Subject must have  care brain CT scan 5. Visualacuity/hearing adequate Chart,  for testing Inter- view 6. Fluency inEnglish or Spanish Chart, Test battery or person- Inter- nelavailability view 7. Ability to provide informed Inter-  consent viewExclusion Criteria 1. Significant polytrauma that Chart Significant body would interfere with follow-up trauma may con-  and outcome assessmentfound TBI outcomes testing. 2. Prisoners or patients in custody Chart,Inter- view 3. Pregnancy in female subjects Chart, Inter- view 4.Patients on psychiatric hold Chart  (e.g., 5150, 5250) 5. Majordebilitating baseline Chart, Debilitating psychiatric  mental healthdisorders (e.g., Inter- disorders can significantly  schizophrenia orbipolar dis- impact the reliability of  order) that would interfere withfollow up and/or pose  follow-up and the validity of difficulties inattributing to  outcome assessment index TBI. 6. Major debilitatingneurological Chart, Documented debilitating  disease (e.g., stroke, CVA,Inter- baseline cognitive impair-  dementia, tumor) impairing view mentwill confound  baseline awareness cognition outcome assessment in  orvalidity of follow-up and addition to not  outcome assessment beingfully consentable. 7. Significant history of pre- Chart,  existingconditions that would Inter-  interfere with follow-up and view  outcomeassessment (e.g.,  substance abuse, alcoholism,  HIV/AIDS, majortransmittable  diseases that may interfere with  consent, end-stagecancers,  learning disabilities, develop-  mental disorders) 8.Contraindications to MRI (for MRI  CA + MRI cohort) Screen- ing 9. Lowlikelihood of follow-up Inter-  (e.g., participant or family view indicating low interest, residence  in another state or country, homelessness or lack of reliable  contacts) 10. Current participant inan Chart, Exception to co-enroll-   interventional trial (e.g., Inter-ment exclusion is made   drug, device, behavioral) view for sitesparticipating in Resuscitation Outcomes Consortium PrehospitalTranexamic Acid for TBI Study. 11. Penetrating TBI Chart 12. Spinal cordinjury with ASIA Chart   score of C or worse

For each of the 3 clinical groups (i.e., ED, ADM, and ICU), the subjectswere further placed into one of three different assessment cohorts:Brief Assessment (BA Cohort), Compressive Assessment (CA) Cohort, orComprehensive Assessment+MRI (CA+MRI) Cohort. See Table 3 for Milestoneplan with 80% follow up rate.

TABLE 3 (Add) Year 1 Year 2 Year 3 Year 4 Total Group CA + MRI CA N CA +MRI CA N CA BA N BA N ED 150 87 237 50 58 108 155 100 255 300 900 ADM150 87 237 50 58 108 155 100 255 300 900 ICU 150 87 237 50 58 108 155100 255 300 900 Controls 0 99 99 0 66 66 135 0 135 0 300 Totals 450 360810 150 240 390 600 300 900 900 3000

The Brief Assessment (BA) Cohort included 1200 total subjects, with 400subjects each for ED, ADM, and ICU Groups. The following data wasgathered for the BA Cohort: demographic and full clinical course data;blood draw for serum, plasma, DNA and RNA on Day 1 (<24 hours ofinjury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);clinical brain CT scan from Day 1 acquired as part of hospital course;and outcome data collected via structured telephone interview at 2weeks, 3, 6, and 12 months using NIH TBI-CDEs v.2.0 Core outcomemeasures as published on the NINDS CDE website.

The Compressive Assessment (CA) Cohort included 1200 total subjects,with 300 subjects+100 controls each for ED, ADM, and ICU Groups. Thefollowing data was gathered for the CA Cohort: demographic and fullclinical course data; high density daily clinical data for ADM and ICUGroups; blood draw for serum, plasma, RNA, and DNA on Day 1 (<24 hoursof injury); repeat blood draw for serum within 3-6 hours of the Day 1baseline collection (optional for sites to include this component);blood draw for serum, plasma and RNA of Day 3 (48-72 hours) and 5(96-120 hours) for ADM and ICU; collection of cerebrospinal fluid ondays 1 through 5 (optional for sites to include this component); allclinical brain CT scans acquired as part of hospital course; blood drawfor serum, plasma and RNA at 2 weeks and 6 months; and outcome datacollected via structured in-person interview at 2 weeks, 6, and 12months and at 3 months via structured telephone interview using NIHTBI-CDEs v.2.0 Core, Basic and Supplemental outcome measures.

The Comprehensive Assessment+MRI (CA+MRI) Cohort included 600 totalsubjects, with 200 each for ED, ADM, and ICU Groups. The following datawas gathered for the CA+MRI Cohort: demographic and full clinical coursedata; high density daily clinical data for ADM and ICU Groups; blooddraw for serum, plasma, RNA, and DNA on Day 1 (<24 hours of injury);repeat blood draw for serum within 3-6 hours of the Day 1 baselinecollection (optional for sites to include this component); blood drawfor serum, plasma, and RNA on Day 3 (48-72 hours) and 5 (96-120 hours)for ADM and ICU; collection of cerebrospinal fluid on days 1 through 5(optional for sites to include this component); all clinical head CTscans acquired as part of hospital course; blood draw for serum, plasmaand RNA at 2 weeks and 6 months; 3T research MRI acquired at 2 weeks and6 months; and outcome data collected via structured in-person interviewat 2 weeks, 6, and 12 months and at 3 month via structured telephoneinterview using NIH TBI-CDEs v.2.0 Core, Basic, and Supplemental outcomemeasures.

Upon enrollment, data collection began in the hospital. For CA+MRIpatients, the 2-week MRI was completed at 14 days±4 days from the dateof injury. Corresponding 2-week outcomes were completed±3 days of the2-week MRI. For CA and BA patients, 2-week outcomes were completed±4days of 14 days from the date of injury. Outcomes at 3 months werecompleted±7 days of 90 days from the date of injury. For CA+MRIpatients, MRIs at 6 months were completed±14 days of 180 days from thedate of injury, with corresponding 6-month outcomes±14 days of the6-month MRI. For CA and BA patients, 6-month outcomes were completed±14days of 180 days from the date of injury. BTACT should be completed with±7 days of Outcomes (but not on the same day and no greater than 201days from injury). Outcomes at 12 months were completed±30 days of 360days from the date of injury.

HsTnI and UCH-L1 were measured in a small sample size of 59 patientsfrom Study 1 using the Abbott Architect STAT hsTnI assay (Table 4).

TABLE 4 Subject Characteristics by CT Scan and MRI Result Subject TotalCT or MRI Positive* CT and MRI Negative* Characteristics (n = 59) (n =46, 77.97%) (n = 13, 22.03%) P value Age  46.0  45.5  50.0 0.7419 [24.0to 60.0] [23.0 to 60.0] [39.0 to 57.0] Sex Male 50/59 (85%) 39/46 (85%)11/13 (85%) 1.0000 Female 9/59 (15%) 7/46 (15%) 2/13 (15%)Race/Ethnicity African-American or African 6/58 (10%) 4/45 (9%) 2/13(15%) 0.2398 Caucasian 48/50 (83%) 39/45 (87%) 9/13 (69%) Hispanic 4/58(7%) 2/45 (4%) 2/13 (15%) TBI History Yes, with No LOC 9/56 (16%) 3/43(7%) 6/13 (46%) 0.0037 Yes, with LOC 8/56 (14%) 6/43 (14%) 2/13 (15%) NoPrior TBI 39/56 (70%) 34/43 (79%) 5/13 (38%) ED Presentation Loss ofConsciousness No 6/58 (10%) 2/45 (4%) 4/13 (31%) 0.0227 Yes 47/58 (81%)38/45 (84%) 9/13 (69%) Unknown 5/58 (9%) 5/45 (11%) Glasgow Coma Scale 15.0  14.0  15.0 0.0162  [3.0 to 15.0]  [3.0 to 15.0] [15.0 to 15.0]Glasgow Coma Scale Classification Severe (3-8) 16/59 (27%) 16/46 (35%)0.0177 Moderate (9-12) 3/59 (5%) 3/46 (7%) Mild (13-15) 40/59 (68%)27/46 (59%) 13/13 (100%) Mechanism of Injury Motor vehicle(driver/passenger) 10/59 (17%) 9/46 (20%) 1/3 (8%) 0.2975Motercycle/ATV/golf cart 5/59 (8%) 3/46 (7%) 2/13 (15%)(driver/passenger) Individual struck by any type of 3/59 (5%) 2/46 (4%)1/13 (8%) vehicle Fall from a moving object 3/59 (5%) 3/46 (7%)(bike/skateboard/horse/etc.) Fall from stationary object 27/59 (46%)20/46 (43%) 7/13 (54%) (roof/ladder/etc.) Assault 10/59 (17%) 9/46 (20%)1/13 (8%) Struck on head by object, not 1/59 (2%) 1/13 (8%) assault(tree/etc.) Alcohol Level (g/dL)  0.1  0.1  0.0 0.1588 [0.0 to 0.2] [0.0to 0.2] [0.0 to 0.0] Drug Screen Negative 51/59 (86%) 41/46 (89%) 10/13(77%) 0.3567 Positive 8/59 (14%) 5/46 (11%) 3/13 (23%) Biomarker ResultsCollection Time Since Injury 771.0 779.4 743.0 0.7383 (Minutes)(+/−339.8) (+/−296.8) (+/−468.7 ) UCH-L₁ (pg/mL) 342.5 514.0  62.4<0.0001 [102.8 to 718.3] [167.2 to 359.8]  [44.5 to 136.8] PrognosticScores Glasgow Outcome Scale (3 months)  6.0  5.5  7.0 0.0130 [5.0 to7.0] [4.0 to 7.0] [7.0 to 7.0] Glasgow Outcome Scale (6 months)  6.0 6.0  7.0 0.1941 [5.0 to 7.0] [4.0 to 7.0] [5.5 to 7.5] Glasgow OutcomeScale (12 months)  7.0  6.5  7.0 0.4412 [5.0 to 8.0] [5.0 to 8.0] [6.0to 8.0] Rivermead Questionnaire First 3  0.0  0.0  0.0 0.8378 Items (6months) [0.0 to 2.0] [0.0 to 2.5] [0.0 to 2.0] Rivermead QuestionnaireLast 13  9.0  8.5  13.0 0.5449 Items (6 months)  [4.0 to 15.0]  [4.0 to15.0]  [0.0 to 27.0] WAIS-III Processing Speed Index  30.0  30.0  43.00.3235 (6 months)  [5.0 to 55.0]  [5.0 to 50.0] [18.0 to 77.0]Satisfaction with Life Scale  21.5  21.7  20.4 0.6205 (6 months) (+/−6.2)  (+/−5.7)  (+/−8.5) Functional Independence Measure 126.0126.0 126.0 0.2958 (6 months) [125.0 to 126.0] [124.0 to 126.0] [126.0to 126.0] *24 subjects received an MRI Continuous variables arepresented as median [25-75% Inter Quartile Range] and compared usingWilcoxon rank sum test or Mean (+/− SD) and compared using a t-testbased on the distribution of the data. Categorical variables arepresented as number/total (percent) and compared using Chi-Square orFisher's exact test.

In addition to a blood draw within 24 hours of brain injury, eachpatient had an extensive medical evaluation including head CT,neuropsychiatric testing, Glasgow Coma Score (GCS), and many patientsalso had a follow up MRI within 2 weeks of injury. Following ameticulous standardized blood draw protocol and processing, plasmasamples were aliquoted for storage at −80° C., later thawed and tested.Each sample was run in duplicate with the listed results being anaverage of the two runs. FIG. 1 shows that UCH-L1 levels correlated withinjury throughout the first 24 hours after injury (range approximately2-23 hours). Table 4 shows that ethanol (ETOH) levels did not correlatewith biomarker levels (Pearson Correlation=0.023, p-value=0.89) as ETOHconsumption is frequently related to TBI, in particular severe TBI.

Table 5 shows the analysis of data in a small sample size of 191patients from Study 1, specifically, the analysis of the change betweenTime Point 2 and Time Point 1 in hsTnI and UCH-L1 levels. The Time Point1 sample is taken within 24 hours from injury and Time Point 2 sample istaken about 3-6 hours after the Time Point 1 sample is taken. UCH-L1appears to have a significant change from Time Point 1 to Time Point 2(n=191, median delta (i.e., change from Time Point 1 to Time Point2)=−38 pg/mL, Wilcoxon Signed-Rank Test p-value<0.0001). See also FIG.2. FIG. 2 shows a box and whisker plot indicating the UCH-L1 levelsdetermined in all of the patients of the study at Time Point 1 and TimePoint 2. GFAP appears to have a significant change from Time Point 1 toTime Point 2 (n=191, median delta (i.e., change from Time Point 1 toTime Point 2)=−1 pg/mL, Wilcoxon Signed-Rank Test p-value=0.7932). Seealso FIG. 34. FIG. 34 shows a box and whisker plot indicating the GFAPlevels determined in all of the patients of the study at Time Point 1and Time Point 2. HsTnI appears to have a significant change from TimePoint 1 to Time Point 2 (n=89, median delta (i.e., change from TimePoint 1 to Time Point 2)=0.091537 pg/mL, Wilcoxon Signed-Rank Testp-value<0.6085). See also FIG. 3. FIG. 3 shows a box and whisker plotindicating the hsTnI levels determined in all of the patients of thestudy at Time Point 1 and Time Point 2. These data were simply analysescomparing the time points, and are not based on CT, MRI or GCS+/−.UCH-L1 appears to have a significant change from Time Point 1 to TimePoint 2 (n=191, median delta (i.e., change from Time Point 1 to TimePoint 2)=−38 pg/mL, Wilcoxon Signed-Rank Test p-value<0.0001).

TABLE 5 Sample Wilcoxon Size Minimum Maximum Signed- Assay (n) MedianDelta Delta Delta Rank Statistic P value hsTnI 89 0.091537 −226.483729419.421 122 0.6085 UCH-L1 191 −38 −6070 2528 −5398.5 <0.0001 GFAP 191−1 −18183 10368 193.5 0.7932

The data from the 191 samples from Study 1 was further analyzed based onwhen the Time Point 1 sample was taken, e.g., 0 to about 6 hours afterinjury (“0-6 hour group”), about 6 to about 12 hours after injury (“6-12hour group”) and correlated with head CT scan results and/or GCS scores.The hsTnI levels in the Time Point 1 and Time Point 2 samples werecompared to the positive or negative head CT scan results and/or GCSscores indicating mild or moderate/severe TBI. As this analysis wasbased on the time of when the Time Point 1 sample was taken, the numberof subjects per group was small. See Table 6.

TABLE 6 Time Range (hours)* Total # of 0-6 6-12 12-18 18-24 >24 SubjectsCT Scan Positive 6 21 33 31 4 95 Negative 8 17 25 25 0 75 GCS Mild 12 2745 41 1 126 Score Moderate/ 2 10 15 15 2 44 Severe *time of first sampletaken from time of injury

CT Scan. FIG. 4 shows the hsTnI results in the subjects from the 0-6hour group that had positive or negative head CT scans. FIG. 4 shows thedistribution of the hsTnI levels at Time Point 1 and Time Point 2 in thesubjects that had a positive or negative CT scan. The hsTnI levels inthe CT positive subjects were higher than the CT negative subjects atboth Time Point 1 and Time Point 2 for the 0-6 hour group. The hsTnIlevels in the subjects from the 0-6 hour group having a positive CT scanhad a striking increase from Time Point 1 to Time Point 2.

FIG. 5 shows the absolute amount (“absolute delta”) in hsTnI levels insubjects from the 0-6 hour group. Subjects having a positive head CTscan had a greater change in hsTnI levels from Time Point 1 to TimePoint 2 compared to subjects having a negative head CT scan.

GCS Scores. FIG. 6 shows the hsTnI results in the subjects from the 0-6hour group that were identified as having mild or moderate to severe(“moderate/severe”) TBI based on their GCS score. FIG. 6 shows thedistribution of the hsTnI levels at Time Point 1 and Time Point 2 in thesubjects determined to have mild or moderate/severe. The hsTnI levels inthe moderate/severe subjects were significantly higher than the mildsubjects at both Time Point 1 and Time Point 2 for the 0-6 hour group.The hsTnI levels in the 0-6 hour group had a striking increase from TimePoint 1 to Time Point 2.

FIG. 7 shows the absolute amount (or change between Time Point 1 andTime Point 2; “absolute delta”) in hsTnI levels of 0-6 hour group.Subjects determined to have moderate/severe TBI based on the GCS scorehad a larger change in hsTnI levels from Time Point 1 to Time Point 2compared to subjects determined to have mild TBI based on the GCS score.

FIG. 8 shows ROC analysis of UCH-L1 assay results at Time Point 1 (takenwithin 0 to 12 hours after head injury) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 9 shows ROC analysis of UCH-L1 assay results at Time Point 1 (takenmore than 12 hours after head injury) correlated with mild vs.moderate/severe TBI GCS scores.

FIG. 10 shows ROC analysis of absolute amount (“absolute delta”) of thecombination of hsTnI levels and UCH-L1 levels (i.e., the absolutedifference between hsTnI levels at Time Point 2 and hsTnI levels at TimePoint 1 and the absolute difference between UCH-L1 levels at Time Point2 and UCH-L1 levels at Time Point 1) correlated with GCS Score result(mild vs. moderate/severe). The sample at Time Point 1 is taken within 0to 12 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken. FIG. 11shows ROC analysis of absolute amount (“absolute delta”) of thecombination of hsTnI levels and UCH-L1 levels (i.e., the absolutedifference between hsTnI levels at Time Point 2 and hsTnI levels at TimePoint 1 and the absolute difference between UCH-L1 levels at Time Point2 and UCH-L1 levels at Time Point 1) correlated with GCS Score result(mild vs. moderate/severe). The sample at Time Point 1 is taken morethan 12 hours of head injury while the sample at Time Point 2 is takenabout 3 to about 6 hours after the Time Point 1 sample is taken.

Example 4 Study 2—Development of a Multi-Modality Classification Schemefor Traumatic Brain Injury

The goal of this study was to develop a classification scheme for braininjury that indicates the nature (type) and severity of injury. Forexample, serum biomarkers revealed cell type. Trauma patients weredivided into three groups for analysis: only brain injured, onlynon-brain injured, and combined injury. Brain injured and non-braininjured trauma groups were compared to each other and to the combinationof brain/nonbrain trauma. These trauma groups were compared tonon-trauma controls. CSF from trauma patients was compared to CSF fromnon-trauma patients. A secondary goal was to determine whether any ofthe measures, alone or in combination, had utility as a predictor ofclinical outcome after TBI.

An objective multi-modality classification scheme and outcome measurefor traumatic brain injury was developed based on several measures: 1)blood-based biomarkers; 2) physiologic measures and evaluation; and 3)radiographic measures (CT and 3T MRI). Blood-based biomarkers canindicate which cell types are damaged (e.g., glial vs. neuronal) andradiography can detect structural changes.

Study Site: Trauma patient were recruited at Hennepin County MedicalCenter (HCMC) in the state of Minnesota. Participants included traumapatients of all ages presenting to the HCMC Emergency Department (ED),trauma bay, or as direct transfer to neurosurgery. Trauma patients wereexcluded if they had a major psychiatric or neurologic disorder, weredevelopmentally abnormal, or were prisoners. Subjects were identified bysearching medical records for all trauma admissions and cross-checkingwith the American College of Surgeons trauma registry utilized in thehospital.

All trauma patients were recruited for screening at the time ofpresentation and underwent: 1) a standardized (templated) history andphysical examination; 2) analysis of serum biomarkers if blood is drawnfor other indications; 3) radiographic study as clinically indicated; 4)follow-up as clinically indicated with 1)-3); 5) pathologic specimenanalysis in patients going to the operating room only; 6) CSF analysisin patients receiving ventriculostomy catheters only; 7) brain tissueoxygenation analysis in patients receiving Licox only; and 8) outcomeassessment in the TBI center as clinically indicated. At the time ofadmission, the potential participants underwent a 24 hour screeningprocess (Table 7) before providing informed consent.

TABLE 7 Screening Assessments Adult Pediatric All Surgery-Trauma Historyand Physical Selections from Neurosurgery-Trauma History and PhysicalAwake SCAT3: SAC, Child SCAT3: SSS-C SAC-C, SSS-C OR Pathogenic SpecimenVC CSF Analysis Licox Brain Tissue Oxygenation OR: Patients going to theoperating room; VC: Patients receiving ventriculostomy catheters; Lkox:Patients receiving Licox

In addition, patients who consented and controls (age and gendermatched) undergone the above plus the following additional studies: 1genomic, serum, and CSF; and 2) 3T MRI in select circumstances (serummarkers in the test group; normal markers in the control group). Traumapatients included the full spectrum ranging from non-brain injured,CT-negative to structurally brain injured, requiring surgery. A patientsand controls were recruited over approximately 15 months. Survivingtrauma subjects were followed until they were discharged from HCMCservices. Subjects evaluated in the ER and released were invited forresearch follow-up.

The screening process included a standardized and templated medicalhistory and physical examination. The template was the current“Surgery-Trauma History and Physical” template in EPIC with oneadditional question that asks if the patient suffered a head trauma. Ifthe patient did, three sections automatically dropped down foradditional information. The first was information from the Neurosurgerytrauma history and physical template, including subarachnoid grade,hemorrhage grade, intracerebral hemorrhage, and social history (level ofeducation, employment, living arrangements, and ethnicity). The finaltwo were standardized brain injury assessment tools: the StandardizedAssessment of Concussion (SAC) and the Symptom Severity Score (SSS).Pediatric versions of these assessments were available and used whenindicated. Additionally, the loss of consciousness question alreadyincluded in the “Surgery Trauma History and Physical” template wascopied into this drop down section with a subset of questions thatprovided a more clear understanding of the loss of consciousness eventand the patient's current orientation. The most clinically accurateassessment taken during the first 24 hours of admission was used forfuture data analysis.

Non-TBI subjects were also included based on the following criteria: bebetween 15 and 50 years of age at the time of enrollment; have similarcharacteristics as the TBI population in terms of gender, age,handedness, educational level, and scanner criteria; and be capable ofsufficiently clear communication and language fluency to allow thesubject to provide written informed consent, or assent with parental orguardian consent for minors, and to complete study assessments forparticipation in all parts of the study. Non-TBI subjects were excludedif they have: been diagnosed of mild TBI within the past 6 months; aprior moderate to severe TBI (GCS<13) within past 10 years; epilepsywith recurring seizures in the past 10 years; drug abuse (exceptmarijuana) in the past 10 years based on DAST-10 screening; alcoholabuse based on AUDIT-C screening; current primary Axis I or IIpsychiatric disorders, except for disorders classified as minor and notexpected to impact study conduct or integrity; a history of brain mass,neurosurgery, stroke, white matter disease, and/or dementia; a knowncognitive dysfunction or structural brain disease/malformation; astructural brain injury on prior neuroimaging findings; been prescribedantipsychotic/antiepileptic medications; been unable (such as due tourgent medical care needs) or unwilling to complete study proceduresaccurately or have any conflict of interest that could affect studyresults, in the opinion of the investigator; or contraindications to MRIscanning, including: a. current or suspected pregnancy per sitepractice; b. other conditions that may constitute a hazard to thesubject during study participation, per investigator; and c. inabilityto comply with any part of the site's MR safety policy.

Specimen Collection and Handling. Up to 40 mL (approximately 3tablespoons) of blood were obtained at each specimen collection. 2 tubesof serum and 2 tubes of plasma were drawn at Encounters 1, 2 4, and 5.At Encounter 3, 2 tubes of serum, 1 tube of plasma and 1 or 2 tubes ofwhole blood were drawn. These study specimens were processed, aliquoted,frozen, and shipped to Abbott Laboratories for biomarker testing andstorage. Sample aliquots were sent to testing sites for additional TBIbiomarker testing. A specimen was considered unevaluable for the studyif: it contained insufficient volume to perform necessary measurements,it was grossly hemolyzed, lipemic, or icteric; it was not collected inthe proper type of collection tube; it was not properly labeled; or itwas not properly stored by the collection site or at AbbottLaboratories.

Serum specimens were obtained via blood draw. If a blood draw wasobtained at the time of admission for clinical purposes, additionalspecimen was obtained and retained for research purposes. If blood wasnot drawn for clinical purposes, trained research personnel drawn theblood required for research. The blood was drawn through venipunctureunless a central venous access was required by the standard of care, inwhich case the blood was withdrawn via that access. The first blood drawwas taken upon admission, the second 3-6 hours after the first, and thethird was taken at 24 hours after the trauma. Discovery efforts werealso ongoing to find genetic markers of susceptibility to TBI orpredictive markers of TBI. At each time point, 40 mL (less than 3tablespoons) of blood was collected: 20 mL of serum (2 tubes) and 20 mLof plasma (2 tubes). During Encounter 1, only 2 tubes of serum and 1tube of plasma were collected for blood biomarker analysis. This wholeblood collection was 6.0 mL in a whole blood tube. If a patient wasenrolled at Encounter 2 instead of Encounter 1, they had 2 tubes ofserum and 1 tube of plasma collected for blood biomarker analysis. Thiswhole blood collection was 6.0 mL in a whole blood tube.

The amount of blood drawn was limited according to the NINOSstandardized table (Table 8). The number of attempts to draw blood waslimited for children under the age of seven to two attempts. In the casethat the NINOS standardized table did not allow enough blood to be drawnfor the study or there were two failed attempts to draw blood in a childpatient, access to leftover blood samples drawn under the clinicalstandard of care was requested for complete biomarker analysis in thisstudy. This blood draw allowed for the analysis of up to 390 blood-basedbiomarkers related to traumatic brain injuries.

TABLE 8 Maximum Allowable Total Blood Draw Volumes Minimum Hgb Maximumrequired at allowable volume Maximum time of blood (mL) in one bloodvolume (clinical + Minimum Hgb draw if subject Body Wt Body Wt Totalblood draw (=2.5% of research) (mL) in required at time has respiratory/(Kg) (lbs) volume (mL) total blood volume) a 30-day period of blood drawCV compromise 1 2.2 100 2.5 5 7.0 9.0-10.0 2 4.4 200 5 10 7.0 9.0-10.0 36.3 240 6 12 7.0 9.0-10.0 4 6.8 320 8 16 7.0 9.0-10.0 5 11 400 10 20 7.09.0-10.0 6 13.2 480 12 24 7.0 9.0-10.0 7 15.4 560 14 28 7.0 9.0-10.0 817.6 640 16 32 7.0 9.0-10.0 9 19.8 720 18 36 7.0 9.0-10.0 10 22 800 2040 7.0 9.0-10.0 11-15 24-33  880-1200 22-30 44-60 7.0 9.0-10.0 16-2035-44 1280-1600 32-40 64-80 7.0 9.0-10.0 21-25 46-55 1680-2000 42-50 64-100 7.0 9.0-10.0 26-30 57-66 2080-2400 52-60 104-120 7.0 9.0-10.031-35 68-77 2480-2800 62-70 124-140 7.0 9.0-10.0 36-40 79-88 2880-320072-80 144-160 7.0 9.0-10.0 41-45 90-99 3280-3600 82-90 164-180 7.09.0-10.0 46-50 101-110 3680-4000  92-100 184-200 7.0 9.0-10.0 51-55112-121 4080-4400 102-110 204-220 7.0 9.0-10.0 56-60 123-132 4480-4800112-120 224-240 7.0 9.0-10.0 61-65 134-143 4880-5200 122-130 244-260 7.09.0-10.0 68-70 145-154 5280-5600 132-140 264-280 7.0 9.0-10.0 71-75156-165 5680-6000 142-150 284-300 7.0 9.0-10.0 76-80 167-176 6080-6400152-160 304-360 7.0 9.0-10.0 81-85 178-187 6480-6800 162-170 324-340 7.09.0-10.0 86-90 189-198 6880-7200 172-180 344-360 7.0 9.0-10.0 91-95200-209 7280-7600 182-190 364-380 7.0 9.0-10.0  96-100 211-220 7680-8000192-200 384-400 7.0 9.0-10.0

In addition to the initial physical exam, those patients that were sentto the operating room undergone pathologic specimen analysis, thosepatients that received Licox had brain tissue oxygenation informationrecorded, and those patients that received ventriculostomy catheters hadCSF collected for analysis. In order to analyze the CSF, 5.0 mL wascollected at the same intervals that blood was drawn. Radiographicstudies were performed in accordance with the standard of care. None ofthe assessments performed during the screening processes were analyzedwith the rest of the data until informed consent was obtained. If thepatient did not ultimately consent to research, the specimens, andinitial assessments were discarded.

After the participants were discharged, the patients' medical recordswere accessed for information about the clinical course, including timespent in the ED, any surgeries or other neuromonitoring methods used,and the acute care outcome evaluation. If the patient spent time in theICU, information was extracted from that time period as well, includingdata from Moberg monitors and daily therapeutic intensity level.

Trauma patients were divided into three groups for analysis: only braininjured, only non-brain injured, and combined injury. Two age- andgender-matched control groups were included in this study and recruitedfrom the ED: non-trauma and CSF controls. Non-trauma controls were thosewho did not experience any trauma, and this group composed largely offamily and friends of the patients admitted for brain injury. Bothcontrol groups were consented to undergo a single intensive assessmentthat included a blood draw and cognitive, neurological, and quality oflife assessments (SAC, NOS-TBI, QoLABI). Patients receiving electiveventriculostomy or lumbar drain catheters were (pre-operatively)consented to be a part of the CSF control group. 5 mL of CSF wascollected from the ventriculostomy catheter of patients in this controlgroup for comparison with the CSF collected from the portion of thestudy group that received a ventriculostomy catheter as a part of theirstandard of care. The CSF control group was also offered the chance toparticipate in the same intensive assessment as the other two controlsgroups that included a blood draw and a 3T MRI scan.

Follow-Up: All patients that consented to participate in the follow-upportion of the study were asked to return to the hospital. Patients whoreturned were seen in the TBI Outpatient Clinic at 2 weeks, 4 weeks,months, 6 months, and 1 year. If they did not have a scheduledappointment at the TBI Outpatient Clinic, they were scheduled a time tocome into the Brain Injury Research Lab (PL.610) at those time points.Table 9 provides a timeline for each of the assessments. Blood draws forbiomarker analysis were done at each of the five follow-up time pointsin the same method, as described above. The outcome assessment batterylisted in Tables 10 and 11 were completed at 3 months, 6 months, and oneyear. Radiographic scans that were a part of the standard of care wereaccessed through the participant's medical records, but select consentedparticipants and controls also underwent 3T MRI scans at 2 weeks and 6months after their brain injury. Each MRI examination took approximatelyone hour and included the following pulse sequences: (1) Sagittal shortTR localizer, (2) Axial Fse, (3) Axial FLAIR, (4) Axial SWI, (5) AxialT2* imaging. In the case that patients were not able to come into thehospital for follow-up, they were contacted via phone at three monthsand one year after their injury to complete the BT ACT, which was a15-20 minute cognitive assessment designed to be administered over thephone.

TABLE 9 Outcome Timeline Blood 3T CSF CT Assess- Total Time Draw MRICollection Scan ments (minutes) ADM X X  10 (if indicated) 2 weeks X X 70 4 weeks X  10 3 months X X  70 6 months X X  70 1 year X X X* X 160(130 without CT)

TABLE 10 Outcome Assessments-Not Finalized Adult Pediatric Outcome AllGOS and GOSE Pediatric GOSE Awake SCAT3; SSS Child SCAT3: Child & andSAC Parent Report; SAC-C GOAT COAT Duration of Amnesia Duration ofAmnesia NOS-TBI NOS-TBI Quality of Life Quality of Life; MPA-4Neuropsydtiatric Rating Schedule Pediathric Quality of Life Inventory(For Child and for Proxy) Not CRS-R (Just Brain Stem Reflex Grid?) Awake

TABLE 11 Possible Infant Assessments Name Age Time Description BayleyIII, 0-3.5 30-90 Cognitive, language (receptive and BSID* expressive)and motor development Most commonly used in test for this age rangeBITSEA* 1-3 7-12 Parent perception of Social and Emotional behavior 17Items of 42 are for autism, so may be able to be made shorter CBCL 1.5-525-30 Parent perception of performance on Activities, Social, and Schoolperformance MSEL* 1 15 Cognitive and Motor Ability 3 25-35 (Gross Motor,Visual Reception, 5 35-40 fine Motor, Language 60 Mostly for readinessfor school Shape School* 3-6 45-75 Inhibition and Switching Processes:Emerging Executive Functions Trails- 2-6 5-10 NeuropsychologicalFuncton: Preschool* Psychomotor speed, Conplex Attention, Executivefunction Advanced Trail making Test *Requested Access

Statistical Analysis Plan. Biomarker data was analyzed by examiningmaximum concentration draw for each biomarker per patient, or by timefrom incident buckets, or both. To address the primary objective ofdetermining associations between biomarker concentrations in blood andclinical neurological and magnetic resonance imaging data, multipleanalyses was employed. Principal components analysis was used to examinewhich biomarkers may be explaining the same variance, or if a biomarkerwas contributing very little variance. The biomarkers were used in alogistic regression analysis, and some biomarkers were excluded based onthe results from the principal components analysis, and clinical input.A significance level of 0.05 was used for the logistic regressionanalysis. ROC analysis was also used to examine the predictive abilityof each biomarker in determining MRI status or neurological testingoutcomes, for this set of data.

Example 5 Study 2—Analysis

High sensitivity troponin I (hsTnI) was measured using the AbbottArchitect STAT hsTnI assay in samples taken from subjects, as describedin Example 4. FIGS. 12, 14, and 16 show that hsTnI (FIG. 12), UCH-L1(FIG. 14), and GFAP (FIG. 15) levels correlated with injury throughoutthe first 24 hours after injury (range approximately 2-23 hours) basedon CT scan results. FIGS. 13, 15, and 17 show that hsTnI (FIG. 13),UCH-L1 (FIG. 15), and GFAP (FIG. 17) levels correlated with injurythroughout the first 24 hours after injury (range approximately 2-23hours) based on GCS score.

Samples Taken from Subjects within 2 hours of Injury: hsTnI levels andUCH-L1 in samples taken from human subjects within about 2 hours ofsuspected injury were measured. FIG. 18 shows ROC analysis of hsTnIlevels correlated with CT status (positive vs. negative CT scan result;AUC=0.430). Table 12 shows the sensitivities and specificities of usinghsTnI cut-off levels to predict a positive CT scan result.

TABLE12 CT Scan-Single Biomarker Reference Levels Analysis LevelsSensitivity Specificity Biomarker (pg/mL) (%) (%) hsTnI 1.15 87.5 31.251.29 75.0 31.25 UCH-L1 854 83.33 69.57 688 91.67 68.12 78 91.67 30.43GFAP 459 66.67 98.55 193 75.00 97.10 181.9 83.33 97.10 108 91.67 91.3034 91.67 71.01 33 100.00 68.12 30 100.00 66.67 11 100.00 31.88

FIG. 19 shows ROC analysis of hsTnI correlated with GCS Score results(mild vs. moderate/severe TBI; AUC=0.588). Table 13 shows thesensitivities and specificities of using hsTnI cut-off levels to predictmoderate/severe TBI based on GCS scores.

TABLE 13 GCS Score-Single Biomarker Reference Levels Analysis LevelsSensitivity Specificity Biomarker (pg/mL) (%) (%) hsTnI 5.80 85.71 33.334.71 85.71 40.0 UCH-L1 3019 70.00 85.92 2919 80.00 84.51 856 90.00 69.01305 100.00 54.93 78 100.00 30.99 GFAP 36 70.00 69.01 28 80.00 60.56 2390.00 59.16 12 90.00 35.21 11 90.00 29.58

Samples Taken from All Subjects: FIG. 20 shows the ROC curve correlatingthe hsTnI levels for all of the subjects at Time Point 1 with CT scanresult. FIG. 26 shows the ROC curve correlating the UCH-L1 levels forall of the subjects at Time Point 1 with CT scan result. FIG. 30 showsthe ROC curve correlating the GFAP levels for all of the subjects atTime Point 1 with CT scan result. The sensitivity and specificity ofreference levels for all of the subjects based on the ROC curve is shownin Table 14.

TABLE 14 CT Scan-Single Biomarker Reference Levels Analysis ReferenceSensitivity Specificity Biomarker Level (pg/mL) (%) (%) hsTnI 1.65 81.5820.59 2.16 71.05 30.88 14.75 31.58 79.41 30.43 21.05 82.35 UCH-L1 278331.58 75.00 413 81.58 45.59 383 84.21 41.18 340 86.84 39.71 293 86.8430.88 GFAP 1929 31.58 100.00 164 81.58 85.29 132 84.21 82.35 116 86.8477.94 109 92.11 76.47 108 94.74 76.47 33 100.00 38.24 27 100.00 30.88

FIG. 28 shows ROC analysis of the combination of hsTnI levels and UCH-L1levels correlated with CT status (positive vs. negative CT scan result)in samples taken at a first time point 1 within 24 hours after headinjury. Table 15 shows the sensitivities and specificities of using acombination of hsTnI cut-off levels and UCH-L1 cut-off levels to predicta positive CT scan result. The cutoffs in the scenario below are wherethe subject is above the cutoff for either hsTnI or UCH-L1 (or both).

TABLE 15 CT Scan-Both Biomarkers Reference Levels Analysis hsTnI LevelsUCH-L1 Levels Sensitivity Specificity (pg/mL) (pg/mL) (%) (%) 10 45076.32 32.35 10 400 81.58 29.41  5 550 81.58 29.41

FIG. 32 shows ROC analysis of the combination of hsTnI levels and GFAPlevels correlated with CT status (positive vs. negative CT scan result)in samples taken at a first time point 1 within 24 hours after headinjury. Table 16 shows the sensitivities and specificities of using acombination of hsTnI cut-off levels and GFAP cut-off levels to predict apositive CT scan result. The cutoffs in the scenario below are where thesubject is above the cutoff for either hsTnI or GFAP (or both).

TABLE 16 CT Scan-Both Biomarkers Reference Levels Analysis hsTnI LevelsGFAP Levels Sensitivity Specificity (pg/mL) (pg/mL) (%) (%)  5 150 86.8454.41 10 150 84.21 64.71 10 100 94.74 58.82 15 100 94.74 64.71 15  5094.74 42.65 15 100 94.74 64.71 15 150 84.21 70.59 20 150 84.21 70.59 35150 81.58 73.53 50 150 81.58 82.53 50 100 94.74 75.00

FIG. 21 shows the ROC curve correlating the hsTnI levels for all of thesubjects at Time Point 1 with GCS scores. FIG. 27 shows the ROC curvecorrelating the UCH-L1 levels for all of the subjects at Time Point 1with GCS scores. FIG. 31 shows the ROC curve correlating the GFAP levelsfor all of the subjects at Time Point 1 with GCS scores. The sensitivityand specificity of reference levels for all of the subjects based on theROC curve is shown in Table 17.

TABLE 17 GCS Score-Single Biomarker Reference Levels Analysis ReferenceLevel Sensitivity Specificity Biomarker (pg/mL) (%) (%) hsTnI 43.7932.14 94.87 21.23 46.43 85.90 1.94 85.71 30.77 UCH-L1 5895 32.14 96.15515 82.14 50.00 410 85.71 43.59 305 89.29 34.62 293 89.29 30.77 GFAP2972 32.14 98.72 52 82.14 42.31 47 85.71 39.74 34 89.29 33.33

FIG. 29 shows ROC analysis of the combination of hsTnI levels and UCH-L1levels correlated with GCS Score result (mild vs. moderate/severe) insamples taken at a first time point 1 within 24 hours after head injury.Table 18 shows the sensitivities and specificities of using acombination of both hsTnI cut-off levels and UCH-L1 cut-off levels topredict moderate/severe TBI based on GCS scores. The cutoffs in thescenario below are where the subject is above the cutoff for eitherhsTnI or UCH-L1 (or both).

TABLE 18 GCS Score-Both Biomarkers Reference Levels Analysis hsTnILevels UCH-L1 Levels Sensitivity Specificity (pg/mL) (pg/mL) (%) (%) 10400 89.29 30.77 10 550 85.71 39.74  5 550 89.29 30.77  5 500 89.29 29.49

FIG. 33 shows ROC analysis of the combination of hsTnI levels and GFAPlevels correlated with GCS Score result (mild vs. moderate/severe) insamples taken at a first time point 1 within 24 hours after head injury.Table 19 shows the sensitivities and specificities of using acombination of both hsTnI cut-off levels and GFAP cut-off levels topredict moderate/severe TBI based on GCS scores. The cutoffs in thescenario below are where the subject is above the cutoff for eitherhsTnI or GFAP (or both).

TABLE 19 GCS Score-Both Biomarkers Reference Levels Analysis hsTnILevels GFAP Levels Sensitivity Specificity (pg/mL) (pg/mL) (%) (%)  5 70 85.71 32.05 10  70 85.71 39.74 10 100 85.71 48.72 15 100 85.71 53.8515 150 82.14 62.82 20 100 85.71 53.85 20 150 82.14 62.82 35 100 85.7156.41 35 150 82.14 66.67 50 150 78.57 73.08

Example 6 Study 2—Absolute Amount Analysis

Samples Taken from Subjects within 2 hours of Injury: hsTnI levels weremeasured in samples taken from human subjects at a first time pointwithin about 2 hours of suspected injury and samples taken from thehuman subjects at a second time point 3-6 hours after the first timepoint. FIG. 22 shows ROC analysis of absolute amount (“absolute delta”)of hsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with CT status(positive vs. negative CT scan result; AUC=0.514). Table 20 shows thesensitivities and specificities of using hsTnI cut-off levels to predicta positive CT scan result, wherein a value less than the cutoff ispredicted to have a positive CT scan result.

TABLE 20 CT Scan-hsTnI Absolute Delta Analysis Absolute AmountSensitivity Specificity Biomarker (pg/mL) (%) (%) hsTnI 2.54 66.67 55.561.16 83.33 27.78

FIG. 23 shows ROC analysis of absolute amount (“absolute delta”) ofhsTnI results (i.e., the absolute difference between hsTnI levels atTime Point 2 and hsTnI levels at Time Point 1) correlated with GCS Scoreresult (mild vs. moderate/severe; AUC=0.380). Table 21 shows thesensitivities and specificities of using hsTnI cut-off levels to predictmoderate/severe TBI based on GCS scores, wherein a value less than thecutoff is predicted to have moderate/severe TBI.

TABLE 21 GCS Score-hsTnI Absolute Delta Analysis Absolute AmountSensitivity Specificity Biomarker (pg/mL) (%) (%) hsTnI  4.73 66.6731.25 20.62 83.33 12.5

Samples Taken from All Subjects: FIG. 24 shows the ROC curve correlatingthe hsTnI levels for all of the subjects at Time Point 1 with CT scanresult. The sensitivity and specificity of absolute amounts (or changebetween Time Point 1 and Time Point 2) in hsTnI levels for all of thesubjects based on the ROC curve are shown in Table 22.

TABLE 22 Absolute amount Sensitivity Specificity (pg/mL) (%) (%) 16.05088.24 19.36 10.755 82.35 19.36  0.720 35.29 70.97  0.389 29.41 90.32

FIG. 25 shows a ROC curve correlating the absolute amount of change inhsTnI levels for all of the subjects at Time Point 1 and Time Point 2with GCS scores (mild vs. moderate/severe). The sensitivity andspecificity of the absolute amounts (or change between Time Point 1 andTime Point 2) in hsTnI levels for all of the subjects based on the ROCcurve is shown in Table 23.

TABLE 23 Absolute amount Sensitivity Specificity (pg/mL) (%) (%)  3.3345.46 32.43  5.80 72.73 21.62 17.17 81.82 13.51

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the disclosure, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the disclosure, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the disclosure are setout in the following numbered clauses:

Clause 1. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising: a)performing an assay on a sample obtained from the subject within about24 hours after a suspected injury to the head to measure or detect alevel of cardiac troponin I (cTnI) and a level of an early biomarker,wherein the sample is a biological sample and early biomarker comprisesubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillaryacidic protein (GFAP), or a combination thereof; and b) determiningwhether the subject has sustained a mild or a moderate, severe, ormoderate to severe traumatic brain injury (TBI), wherein the subject isdetermined as having (1) a moderate, severe, or a moderate to severetraumatic brain injury when the level of cTnI in the sample is higherthan a reference level of cTnI and the level of the early biomarker inthe sample is higher than a reference level of the early biomarker or(2) a mild traumatic brain injury when the level of cTnI in the sampleis lower than a reference level of cTnI and/or the level of the earlybiomarker in the sample is lower than a reference level of the earlybiomarker.

Clause 2. The method of clause 1, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 3. The method of clause 2, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 4. The method of clause 3, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having amoderate, severe, or a moderate to severe traumatic brain injury.

Clause 5. The method of clause 4, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 6. The method of clause 2, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 7. The method of clause 6, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having mildtraumatic brain injury.

Clause 8. The method of clause 7, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 9. The method of any one of clauses 1 to 8, wherein the referencelevel for cTnI is about 5 pg/mL, about 10 pg/mL, about 15 pg/mL, about20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 10. The method of any one of clauses 1 to 9, wherein thereference level for UCH-L1 is about 400 pg/mL, about 500 pg/mL, or about550 pg/mL.

Clause 11. The method of any one of clauses 1 to 9, wherein thereference level for GFAP is about 70 pg/mL, about 100 pg/mL, or about150 pg/mL.

Clause 12. The method of any one of clauses 1 to 11, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; (c) betweenat least about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or between at least about 1 pg/mL to about1000 pg/mL.

Clause 13. The method of any one of clauses 1 to 12, wherein the sampleis taken within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the suspected injury to the head.

Clause 14. The method of any one of clauses 1 to 13, further comprisingtreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 15. The method of any one of clauses 1 to 14, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 16. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained injury to the head, the methodcomprising: a) performing an assay on a sample obtained from the subjectwithin about 24 hours after a suspected injury to the head to measure ordetect a level of cardiac troponin I (cTnI) and a level of an earlybiomarker in the sample, wherein the sample is a biological sample andearly biomarker comprises ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof; and b) performing a CT scan on the subject when the level ofcTnI in the sample is higher than a reference level of cTnI and thelevel of the early biomarker in the sample is higher than a referencelevel of the early biomarker and not performing a CT scan on the subjectwhen the level of cTnI in the sample is lower than a reference level ofcTnI and/or the level of the early biomarker in the sample is lower thana reference level of the early biomarker.

Clause 17. The method of clause 16, wherein the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the suspected injury to the head.

Clause 18. The method of clause 16 or 17, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 19. The method of clause 18, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 20. The method of any one of clauses 16 to 19, wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography.

Clause 21. The method of clause 20, wherein the references level arecorrelated with control subjects that have not sustained a head injury.

Clause 22. The method of any one of clauses 16 to 21, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 23. The method of any one of clauses 16 to 22, wherein thereference level for UCH-L1 is about 400 pg/mL, about 450 pg/mL, or about550 pg/mL.

Clause 24. The method of any one of clauses 16 to 22, wherein thereference level for GFAP is about 50 pg/mL, about 100 pg/mL, or about150.

Clause 25. The method of any one of clauses 16 to 24, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 29% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; (c) between atleast about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or (e) between at least about 1 pg/mL to about1000 pg/mL.

Clause 26. A method of aiding in the diagnosis and evaluation of a humansubject that has sustained or may have sustained an injury to the head,the method comprising: a) performing an assay on a sample obtained fromthe subject within about 2 hours after a suspected injury to the head tomeasure or detect a level of cardiac troponin I (cTnI) and a level of anearly biomarker, wherein the sample is a biological sample and earlybiomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof; and b)determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker.

Clause 27. The method of clause 26, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 28. The method of clause 27, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 29. The method of clause 28, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having amoderate, severe, or a moderate to severe traumatic brain injury.

Clause 30. The method of clause 29, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 31. The method of clause 27, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 32. The method of clause 31, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having mildtraumatic brain injury.

Clause 33. The method of clause 32, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 34. The method of any one of clauses 26 to 33, wherein the sampleis taken within about 5 minutes, within about 10 minutes, within about12 minutes, within about 15 minutes, within about 20 minutes, withinabout 30 minutes, within about 60 minutes, or within about 90 minutesafter a suspected injury to the head.

Clause 35. The method of any one of clauses 26 to 34, further comprisingtreating the subject assessed as having a moderate, severe, or moderateto severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 36. The method of any one of clauses 26 to 35, further comprisingmonitoring the subject assessed as having mild traumatic brain injury.

Clause 37. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained an injury to the head, the methodcomprising: a) performing an assay on a sample obtained from the subjectwithin about 2 hours after a suspected injury to the head to measure ordetect a level of cardiac troponin I (cTnI) and a level of an earlybiomarker in the sample, wherein the sample is a biological sample andearly biomarker comprises ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof; and b) performing a CT scan on the subject when the level ofcTnI in the sample is higher than a reference level of cTnI and thelevel of the early biomarker in the sample is higher than a referencelevel of the early biomarker and not performing a CT scan on the subjectwhen the level of cTnI in the sample is lower than a reference level ofcTnI and/or the level of the early biomarker in the sample is lower thana reference level of the early biomarker.

Clause 38. The method of clause 37, wherein the subject has received aCT scan before or after the assay is performed.

Clause 39. The method of clause 38, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 40. The method of any one of clauses 37 to 39 wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography.

Clause 41. The method of clause 40, wherein the reference levels arecorrelated with control subjects that have not sustained a head injury.

Clause 42. The method of any one of clauses 37 to 41, wherein the sampleis taken within about 5 minutes, within about 10 minutes, within about12 minutes, within about 15 minutes, within about 20 minutes, withinabout 30 minutes, within about 60 minutes, or within about 90 minutesafter a suspected injury to the head.

Clause 43. The method of any one of clauses 1 to 42, wherein measuringthe level of cTnI is done by an immunoassay or clinical chemistry assay.

Clause 44. The method of any one of clauses 1 to 43, wherein measuringthe level of cTnI comprises: A. contacting the sample, eithersimultaneously or sequentially, in any order with: (1) a cTnI-captureantibody, which binds to an epitope on cTnI or cTnI fragment to form acTnI-capture antibody-cTnI antigen complex, and (2) a cTnI-detectionantibody which includes a detectable label and binds to an epitope oncTnI that is not bound by the cTnI-capture antibody, to form a cTnIantigen-cTnI-detection antibody complex, such that a cTnI-captureantibody-cTnI antigen-cTnI-detection antibody complex is formed, and B.measuring the amount or concentration of cTnI in the sample based on thesignal generated by the detectable label in the cTnI-captureantibody-cTnI antigen-cTnI-detection antibody complex.

Clause 45. The method of any one of clauses 1 to 44, wherein the sampleis selected from the group consisting of a whole blood sample, a serumsample, a cerebrospinal fluid sample, and a plasma sample.

Clause 46. The method of any one of clauses 1 to 45, wherein the sampleis obtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

Clause 47. The method of any one of clauses 1 to 45, wherein the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 48. The method of clause 47, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 49. The method of any one of clauses 1 to 45, wherein the sampleis obtained from a subject that suffers from an autoimmune disease, ametabolic disorder, a brain tumor, hypoxia, a virus, meningitis,hydrocephalus or combinations thereof.

Clause 50. The method of any one of clauses 1 to 49, wherein said methodcan be carried out on any subject without regard to factors selectedfrom the group consisting of the subject's clinical condition, thesubject's laboratory values, the subject's classification as sufferingfrom mild, moderate, severe, or a moderate to severe traumatic braininjury, the subject's exhibition of low or high levels of cTnI, and thetiming of any event wherein said subject may have sustained an injury tothe head.

Clause 51. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or suspected injury to the head tomeasure or detect a level of cardiac troponin I (cTnI) and a level of anearly biomarker, wherein the sample is a biological sample and the earlybiomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker.

Clause 52. The method of clause 51, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 53. The method of clause 52, wherein the subject is suspected ashaving moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 54. The method of clause 53, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having amoderate, severe, or moderate to severe traumatic brain injury.

Clause 55. The method of clause 54, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 56. The method of clause 52, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 57. The method of clause 56, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having mildtraumatic brain injury.

Clause 58. The method of clause 57, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 59. The method of any one of clauses 51 to 58, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 60. The method of any one of clauses 51 to 59, wherein thereference level for UCH-L1 is about 400 pg/mL, about 500 pg/mL, or about550 pg/mL.

Clause 61. The method of any one of clauses 51 to 60, wherein thereference level for GFAP is about 70 pg/mL, about 100 pg/mL, or about150 pg/mL.

Clause 62. The method of any one of clauses 51 to 61, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; (c) betweenat least about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or between at least about 1 pg/mL to about1000 pg/mL.

Clause 63. The method of any one of clauses 51 to 62, wherein the sampleis taken within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 64. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained an injury to the head, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after the actual or suspected injury to the head tomeasure or detect a level of cTnI and a level of an early biomarker inthe sample, wherein the sample is a biological sample and earlybiomarker comprises UCH-L1, GFAP, or a combination thereof; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and the level of theearly biomarker in the sample is higher than a reference level of theearly biomarker and not performing a CT scan on the subject when thelevel of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker.

Clause 65. The method of clause 64, wherein the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the suspected injury to the head.

Clause 66. The method of clause 64 or 65, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 67. The method of clause 66, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 68. The method of any one of clauses 64 to 67, wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography.

Clause 69. The method of clause 68, wherein the references level arecorrelated with control subjects that have not sustained a head injury.

Clause 70. The method of any one of clauses 64 to 69, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 71. The method of any one of clauses 64 to 70, wherein thereference level for UCH-L1 is about 400 pg/mL, about 450 pg/mL, or about550 pg/mL.

Clause 72. The method of any one of clauses 64 to 71, wherein thereference level for GFAP is about 50 pg/mL, about 100 pg/mL, or about150.

Clause 73. The method of any one of clauses 64 to 71, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 29% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; (c) between atleast about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or (e) between at least about 1 pg/mL to about1000 pg/mL.

Clause 74. A method for aiding in the diagnosis and evaluation of mildtraumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI and a level of an early biomarker, wherein thesample is a biological sample and the early biomarker comprises UCH-L1,GFAP, or a combination thereof; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe TBI, wherein the subject is determined ashaving (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI and the level of the early biomarker in the sample ishigher than a reference level of the early biomarker or (2) a mildtraumatic brain injury when the level of cTnI in the sample is lowerthan a reference level of cTnI and/or the level of the early biomarkerin the sample is lower than a reference level of the early biomarker.

Clause 75. The method of clause 74, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 76. The method of clause 75, wherein the subject is suspected ashaving a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 77. The method of clause 76, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects havingmoderate, severe, or a moderate to severe traumatic brain injury.

Clause 78. The method of clause 77, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 79. The method of clause 75, wherein the subject is suspected ashaving mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 80. The method of clause 79, wherein the reference levels of thecTnI and of the early biomarker are correlated with subjects having mildtraumatic brain injury.

Clause 81. The method of clause 30, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 82. A method of aiding in the determination of whether to performa head computerized tomography (CT) scan on a human subject that hassustained or may have sustained an injury to the head, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cardiac troponin I (cTnI) and a level of an earlybiomarker in the sample, wherein the sample is a biological sample andearly biomarker comprises ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and the level of theearly biomarker in the sample is higher than a reference level of theearly biomarker and not performing a CT scan on the subject when thelevel of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker.

Clause 83. The method of clause 82, wherein the sample is taken from thesubject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 84. The method of clause 82 or 83, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 85. The method of clause 84, wherein the subject is suspected ofhaving a traumatic brain injury based on the CT scan.

Clause 86. The method of any one of clauses 82 to 85, wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography.

Clause 87. The method of clause 86, wherein the references level arecorrelated with control subjects that have not sustained a head injury.

Clause 88. A method of treating a mild, moderate, severe, or moderate tosevere traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or injury to the head to measure ordetect a level of cTnI and a level of an early biomarker, wherein thesample is a biological sample and the early biomarker comprises UCH-L1,GFAP, or a combination thereof;

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI and the level of the early biomarker in the sample ishigher than a reference level of the early biomarker or (2) a mildtraumatic brain injury when the level of cTnI in the sample is lowerthan a reference level of cTnI and/or the level of the early biomarkerin the sample is lower than a reference level of the early biomarker;and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 89. The method of clause 88, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 90. A method of treating a mild, moderate, severe, or moderate tosevere traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI and a level of an early biomarker, wherein thesample is a biological sample and the early biomarker comprises UCH-L1,GFAP, or a combination thereof;

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 91. The method of clause 90, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 92. The method of any one clauses 51 to 91 further comprising oftreating the subject with at least one cardioprotective therapy.

Clause 93. The method of clause 92, wherein the at least onecardioprotective therapy comprises a beta-blocker, a diuretic, anAngiotensin-Converting Enzyme (ACE) inhibitor, a calcium channelblocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet,an anticlotting agent, an anticoagulation agent or combinations thereof.

Clause 94. The method of any one of clauses 51 to 93, wherein measuringthe level of cTnI is done by an immunoassay or clinical chemistry assay.

Clause 95. The method of any one of clauses 51 to 94, wherein measuringthe level of cTnI comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex,        -   such that a cTnI-capture antibody-cTnI            antigen-cTnI-detection antibody complex is formed, and    -   B. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 96. The method of any one of clauses 51 to 95, wherein the sampleis selected from the group consisting of a whole blood sample, a serumsample, a cerebrospinal fluid sample, and a plasma sample.

Clause 97. The method of any one of clauses 51 to 96, wherein the sampleis obtained after the subject sustained an injury to the head caused byphysical shaking, blunt impact by an external mechanical or other forcethat results in a closed or open head trauma, one or more falls,explosions or blasts or other types of blunt force trauma.

Clause 98. The method of any one of clauses 51 to 97, wherein the sampleis obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 99. The method of clause 98, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 100. The method of any one of clauses 51 to 99, wherein thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

Clause 101. The method of any one of clauses 51 to 100, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or a moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of cTnI,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 102. The method of any one of clauses 51 to 101, wherein thesample is a whole blood sample.

Clause 103. The method of any one of clauses 51 to 101, wherein thesample is a serum sample.

Clause 104. The method of any one of clauses 51 to 101, wherein thesample is a plasma sample.

Clause 105. The method of any one of clauses 102 to 104, wherein theassay is an immunoassay.

Clause 106. The method of any one of clauses 102 to 104, wherein theassay is a clinical chemistry assay.

Clause 107. The method of any one of clauses 102 to 104, wherein theassay is a single molecule detection assay.

Clause 108. A method for evaluating a human subject for mild traumaticbrain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual suspected injury to the head to measureor detect a level of cardiac troponin I (cTnI) and a level of an earlybiomarker, wherein the sample is a biological sample and the earlybiomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker.

Clause 109. The method of clause 108, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 110. The method of clause 109, wherein the subject is suspectedas having moderate, severe, or moderate to severe traumatic brain injurybased on the Glasgow Coma Scale score.

Clause 111. The method of clause 110, wherein the reference levels ofthe cTnI and of the early biomarker are correlated with subjects havinga moderate, severe, or moderate to severe traumatic brain injury.

Clause 112. The method of clause 111, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 113. The method of clause 108, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 114. The method of clause 113, wherein the reference levels ofthe cTnI and of the early biomarker are correlated with subjects havingmild traumatic brain injury.

Clause 115. The method of clause 114, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 116. The method of any one of clauses 108 to 115, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 117. The method of any one of clauses 108 to 116, wherein thereference level for UCH-L1 is about 400 pg/mL, about 500 pg/mL, or about550 pg/mL.

Clause 118. The method of any one of clauses 108 to 117, wherein thereference level for GFAP is about 70 pg/mL, about 100 pg/mL, or about150 pg/mL.

Clause 119. The method of any one of clauses 108 to 118, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; (c) betweenat least about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or between at least about 1 pg/mL to about1000 pg/mL.

Clause 120. The method of any one of clauses 108 to 119, wherein thesample is taken within about 30 minutes, within about 1 hour, withinabout 2 hours, within about 3 hours, within about 4 hours, within about5 hours, within about 6 hours, within about 7 hours, within about 8hours, within about 9 hours, within about 10 hours, within about 11hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the injury or suspected injury to thehead.

Clause 121. A method of determining whether to perform a headcomputerized tomography (CT) scan on a human subject that has an actualor suspected injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an injury or suspected injury to the head tomeasure or detect a level of cTnI and a level of an early biomarker inthe sample, wherein the sample is a biological sample and earlybiomarker comprises UCH-L1, GFAP, or a combination thereof; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and the level of theearly biomarker in the sample is higher than a reference level of theearly biomarker and not performing a CT scan on the subject when thelevel of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker.

Clause 122. The method of clause 121, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual injury or suspected injury to the head.

Clause 123. The method of clause 121 or 122, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 124. The method of clause 123, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 125. The method of any one of clauses 121 to 124, wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography.

Clause 126. The method of clause 125, wherein the references level arecorrelated with control subjects that have not sustained a head injury.

Clause 127. The method of any one of clauses 121 to 126, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.

Clause 128. The method of any one of clauses 121 to 127, wherein thereference level for UCH-L1 is about 400 pg/mL, about 450 pg/mL, or about550 pg/mL.

Clause 129. The method of any one of clauses 121 to 128, wherein thereference level for GFAP is about 50 pg/mL, about 100 pg/mL, or about150.

Clause 130. The method of any one of clauses 121 to 129, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 65% to 100% and a specificity of between at leastabout 29% to 100%; (b) determined by an assay having a sensitivity of atleast about 85% and a specificity of at least about 33%; (c) between atleast about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or (e) between at least about 1 pg/mL to about1000 pg/mL.

Clause 131. A method for evaluating a human subject for mild traumaticbrain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI and a level of an early biomarker, wherein thesample is a biological sample and the early biomarker comprises UCH-L1,GFAP, or a combination thereof; and

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe TBI, wherein the subject is determined ashaving (1) a moderate, severe, or moderate to severe traumatic braininjury when the level of cTnI in the sample is higher than a referencelevel of cTnI and the level of the early biomarker in the sample ishigher than a reference level of the early biomarker or (2) a mildtraumatic brain injury when the level of cTnI in the sample is lowerthan a reference level of cTnI and/or the level of the early biomarkerin the sample is lower than a reference level of the early biomarker.

Clause 132. The method of clause 131, wherein the subject has received aGlasgow Coma Scale score before or after the assay is performed.

Clause 133. The method of clause 132, wherein the subject is suspectedas having a moderate, severe, or a moderate to severe traumatic braininjury based on the Glasgow Coma Scale score.

Clause 134. The method of clause 133, wherein the reference levels ofthe cTnI and of the early biomarker are correlated with subjects havingmoderate, severe, or a moderate to severe traumatic brain injury.

Clause 135. The method of clause 134, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 3-12.

Clause 136. The method of clause 135, wherein the subject is suspectedas having mild traumatic brain injury based on the Glasgow Coma Scalescore.

Clause 137. The method of clause 136, wherein the reference levels ofthe cTnI and of the early biomarker are correlated with subjects havingmild traumatic brain injury.

Clause 138. The method of clause 137, wherein the reference levels arecorrelated with a Glasgow Coma Scale score of 13-15.

Clause 139. A method of determining whether to perform a headcomputerized tomography (CT) scan on a human subject that has sustainedor may have sustained an injury to the head, the method comprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an injury or suspected injury to the head to measureor detect a level of cardiac troponin I (cTnI) and a level of an earlybiomarker in the sample, wherein the sample is a biological sample andearly biomarker comprises ubiquitin carboxy-terminal hydrolase L1(UCH-L1), glial fibrillary acidic protein (GFAP), or a combinationthereof; and

b) performing a CT scan on the subject when the level of cTnI in thesample is higher than a reference level of cTnI and the level of theearly biomarker in the sample is higher than a reference level of theearly biomarker and not performing a CT scan on the subject when thelevel of cTnI in the sample is lower than a reference level of cTnIand/or the level of the early biomarker in the sample is lower than areference level of the early biomarker.

Clause 140. The method of clause 139, wherein the sample is taken fromthe subject within about 30 minutes, within about 1 hour, within about 2hours, within about 3 hours, within about 4 hours, within about 5 hours,within about 6 hours, within about 7 hours, within about 8 hours, withinabout 9 hours, within about 10 hours, within about 11 hours, withinabout 12 hours, within about 13 hours, within about 14 hours, withinabout 15 hours, within about 16 hours, within about 17 hours, withinabout 18 hours, within about 19 hours, within about 20 hours, withinabout 21 hours, within about 22 hours, within about 23 hours, or withinabout 24 hours of the actual or suspected injury to the head.

Clause 141. The method of clause 139 or 140, wherein the subject hasreceived a CT scan before or after the assay is performed.

Clause 142. The method of clause 141, wherein the subject is suspectedof having a traumatic brain injury based on the CT scan.

Clause 143. The method of any one of clauses 139 to 142, wherein thereference levels of the cTnI and of the early biomarker are correlatedwith positive head computed tomography. Clause 144. The method of clause143, wherein the references level are correlated with control subjectsthat have not sustained a head injury.

Clause 145. A method of treating a mild, moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 24 hours after an actual or suspected injury to the head tomeasure or detect a level of cTnI and a level of an early biomarker,wherein the sample is a biological sample and the early biomarkercomprises UCH-L1, GFAP, or a combination thereof;

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 146. The method of clause 145, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 147. A method of treating a mild, moderate, severe, or moderateto severe traumatic brain injury in a human subject, the methodcomprising:

a) performing an assay on a sample obtained from the subject withinabout 2 hours after an actual or suspected injury to the head to measureor detect a level of cTnI and a level of an early biomarker, wherein thesample is a biological sample and the early biomarker comprises UCH-L1,GFAP, or a combination thereof;

b) determining whether the subject has sustained a mild or a moderate,severe, or moderate to severe traumatic brain injury (TBI), wherein thesubject is determined as having (1) a moderate, severe, or a moderate tosevere traumatic brain injury when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker or (2) a mild traumatic brain injury when the level of cTnI inthe sample is lower than a reference level of cTnI and/or the level ofthe early biomarker in the sample is lower than a reference level of theearly biomarker; and

c) treating the subject assessed as having a mild, moderate, severe, ormoderate to severe traumatic brain injury with a traumatic brain injurytreatment.

Clause 148. The method of clause 147, further comprising monitoring thesubject assessed as having a mild, moderate, severe, or moderate tosevere traumatic brain injury.

Clause 149. The method of any one clauses 108 to 148 further comprisingof treating the subject with at least one cardioprotective therapy.

Clause 150. The method of clause 149, wherein the at least onecardioprotective therapy comprises a beta-blocker, a diuretic, anAngiotensin-Converting Enzyme (ACE) inhibitor, a calcium channelblocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet,an anticlotting agent, an anticoagulation agent or combinations thereof.

Clause 151. The method of any one of clauses 108 to 150, whereinmeasuring the level of cTnI is done by an immunoassay or clinicalchemistry assay.

Clause 152. The method of any one of clauses 108 to 151, whereinmeasuring the level of cTnI comprises:

-   -   A. contacting the sample, either simultaneously or sequentially,        in any order with:        -   (1) a cTnI-capture antibody, which binds to an epitope on            cTnI or cTnI fragment to form a cTnI-capture antibody-cTnI            antigen complex, and        -   (2) a cTnI-detection antibody which includes a detectable            label and binds to an epitope on cTnI that is not bound by            the cTnI-capture antibody, to form a cTnI            antigen-cTnI-detection antibody complex,        -   such that a cTnI-capture antibody-cTnI            antigen-cTnI-detection antibody complex is formed, and    -   B. measuring the amount or concentration of cTnI in the sample        based on the signal generated by the detectable label in the        cTnI-capture antibody-cTnI antigen-cTnI-detection antibody        complex.

Clause 153. The method of any one of clauses 108 to 152, wherein thesample is selected from the group consisting of a whole blood sample, aserum sample, a cerebrospinal fluid sample, and a plasma sample.

Clause 154. The method of any one of clauses 108 to 153, wherein thesample is obtained after the subject sustained an injury to the headcaused by physical shaking, blunt impact by an external mechanical orother force that results in a closed or open head trauma, one or morefalls, explosions or blasts or other types of blunt force trauma.

Clause 155. The method of any one of clauses 108 to 153, wherein thesample is obtained after the subject has ingested or been exposed to achemical, toxin or combination of a chemical and toxin.

Clause 156. The method of clause 155, wherein the chemical or toxin isfire, mold, asbestos, a pesticide, an insecticide, an organic solvent, apaint, a glue, a gas, an organic metal, a drug of abuse or one or morecombinations thereof.

Clause 157. The method of any one of clauses 108 to 156, wherein thesample is obtained from a subject that suffers from an autoimmunedisease, a metabolic disorder, a brain tumor, hypoxia, a virus,meningitis, hydrocephalus or combinations thereof.

Clause 158. The method of any one of clauses 108 to 157, wherein saidmethod can be carried out on any subject without regard to factorsselected from the group consisting of the subject's clinical condition,the subject's laboratory values, the subject's classification assuffering from mild, moderate, severe, or a moderate to severe traumaticbrain injury, the subject's exhibition of low or high levels of cTnI,and the timing of any event wherein said subject may have sustained aninjury to the head.

Clause 159. The method of any one of clauses 108 to 158, wherein thesample is a whole blood sample.

Clause 160. The method of any one of clauses 108 to 158, wherein thesample is a serum sample.

Clause 161. The method of any one of clauses 108 to 158, wherein thesample is a plasma sample.

Clause 162. The method of any one of clauses 159 to 161, wherein theassay is an immunoassay.

Clause 163. The method of any one of clauses 159 to 161, wherein theassay is a clinical chemistry assay.

Clause 164. The method of any one of clauses 159 to 161, wherein theassay is a single molecule detection assay.

1.-57. (canceled)
 58. A method comprising: a) performing at least oneassay for cardiac troponin I (cTnI) and an early biomarker in at leastone sample that is whole blood, serum, plasma, cerebrospinal fluid, atissue sample or a bodily fluid obtained from a subject within about 24hours after an actual or suspected injury to the head, wherein the earlybiomarker comprises ubiquitin carboxy-terminal hydrolase L1 (UCH-L1),glial fibrillary acidic protein (GFAP), or a combination thereof; and b)treating the subject for (1) a moderate, severe, or moderate to severetraumatic brain injury (TBI) when the level of cTnI in the sample ishigher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker, or (2) a mild TBI when the level of cTnI in the sample islower than a reference level of cTnI and/or the level of the earlybiomarker in the sample is lower than a reference level of the earlybiomarker.
 59. The method of claim 58, wherein the assay is selectedfrom the group consisting of an immunoassay, a clinical chemistry assay,and a point-of-care assay.
 60. The method of claim 58, wherein the assayis performed using single molecule detection.
 61. The method of claim58, wherein the subject has received a Glasgow Coma Scale score beforeor after the assay is performed, and (i) wherein the reference level iscorrelated with a Glasgow Coma Scale of 3-12; or (ii) wherein thereference level is correlated with a Glasgow Coma Scale score of 13-15.62. The method of claim 58, wherein the reference level for cTnI isabout 5 pg/mL, about 10 pg/mL, about 15 pg/mL, about 20 pg/mL, about 35pg/mL, or about 50 pg/mL.
 63. The method of claim 58, wherein: (i) thereference level for UCH-L1 is about 400 pg/mL, about 500 pg/mL, or about550 pg/mL; or (ii) the reference level for GFAP is about 70 pg/mL, about100 pg/mL, or about 150 pg/mL.
 64. The method of claim 58, wherein thereference level is (a) determined by an assay having a sensitivity ofbetween at least about 85% to 100% and a specificity of between at leastabout 30% to 100%; (b) determined by an assay having a sensitivity of atleast about 87.5% and a specificity of at least about 31%; (c) betweenat least about 1 pg/mL to about 100 pg/mL; (d) between at least about 1pg/mL to about 500 pg/mL; or (e) between at least about 1 pg/mL to about1000 pg/mL.
 65. The method of claim 58, wherein the sample is takenwherein the sample is taken within about 30 minutes, within about 1hour, within about 2 hours, within about 3 hours, within about 4 hours,within about 5 hours, within about 6 hours, within about 7 hours, withinabout 8 hours, within about 9 hours, within about 10 hours, within about11 hours, within about 12 hours, within about 13 hours, within about 14hours, within about 15 hours, within about 16 hours, within about 17hours, within about 18 hours, within about 19 hours, within about 20hours, within about 21 hours, within about 22 hours, within about 23hours, or within about 24 hours of the actual or suspected injury to thehead.
 66. The method of claim 58, wherein a subject with (1) a moderate,severe, or moderate to severe TBI is treated (a) with one or moretherapeutic agents; (b) with one or more surgical procedures; (c) byprotecting the subject's airway; (d) with one or more therapies selectedfrom the group consisting of rehabilitation, physical therapy,occupational therapy, cognitive behavioral therapy, anger management,counseling and any combinations thereof; or (e) with any combination of(a)-(d); or (2) a mild TBI is treated (a′) with rest; (b′) by abstainingfrom physical activities; (c′) by avoiding light; (d′) by wearingprotective eyewear when in light; (e′) with one or more therapeuticagents; or (f′) with any combination of (a′)-(e′).
 67. The method ofclaim 66, wherein the one or more therapeutic agents is at least onecardioprotective therapy, wherein the at least one cardioprotectivetherapy comprises a beta-blocker, a diuretic, an Angiotensin-ConvertingEnzyme (ACE) inhibitor, a calcium channel blocker, a lipid loweringtherapy, a statin, a nitrate, an antiplatelet, an anticlotting agent, ananticoagulation agent or combinations thereof.
 68. A method comprising:a) performing at least one assay for cardiac troponin I (cTnI) and anearly biomarker in at least one sample that is whole blood, serum,plasma, cerebrospinal fluid, a tissue sample or a bodily fluid obtainedfrom a subject within about 24 hours after an actual or suspected injuryto the head, wherein the early biomarker comprises ubiquitincarboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein(GFAP), or a combination thereof; and b) performing a head computedtomography (CT) scan on the subject when the level of cTnI in the sampleis higher than a reference level of cTnI and the level of the earlybiomarker in the sample is higher than a reference level of the earlybiomarker.
 69. The method of claim 68, wherein the method furthercomprises not performing a head CT scan on the subject and treating thesubject for a mild TBI when the level of cTnI in the sample is lowerthan a reference level of cTnI and/or the level of the early biomarkerin the sample is lower than a reference level of the early biomarker.70. The method of claim 68, wherein the method further comprises rulingout the need to perform a head CT scan on the subject when the level ofcTnI in the sample is lower than a reference level of cTnI and/or thelevel of the early biomarker in the sample is lower than a referencelevel of the early biomarker.
 71. The method of claim 68, wherein theassay is selected from the group consisting of an immunoassay, aclinical chemistry assay, and a point-of-care assay.
 72. The method ofclaim 68, wherein the assay is performed using single moleculedetection.
 73. The method of claim 68, wherein the sample is taken fromthe subject wherein the sample is taken within about 30 minutes, withinabout 1 hour, within about 2 hours, within about 3 hours, within about 4hours, within about 5 hours, within about 6 hours, within about 7 hours,within about 8 hours, within about 9 hours, within about 10 hours,within about 11 hours, within about 12 hours, within about 13 hours,within about 14 hours, within about 15 hours, within about 16 hours,within about 17 hours, within about 18 hours, within about 19 hours,within about 20 hours, within about 21 hours, within about 22 hours,within about 23 hours, or within about 24 hours of the actual orsuspected injury to the head.
 74. The method of claim 68, wherein thereference level for cTnI is about 5 pg/mL, about 10 pg/mL, about 15pg/mL, about 20 pg/mL, about 35 pg/mL, or about 50 pg/mL.
 75. The methodof claim 68, wherein: (i) the reference level for UCH-L1 is about 400pg/mL, about 450 pg/mL, or about 550 pg/mL; or (ii) the reference levelfor GFAP is about 50 pg/mL, about 100 pg/mL, or about
 150. 76. Themethod of claim 68, wherein the reference level is (a) determined by anassay having a sensitivity of between at least about 65% to 100% and aspecificity of between at least about 29% to 100%; (b) determined by anassay having a sensitivity of at least about 85% and a specificity of atleast about 33%; (c) between at least about 1 pg/mL to about 100 pg/mL;(d) between at least about 1 pg/mL to about 500 pg/mL; or (e) between atleast about 1 pg/mL to about 1000 pg/mL.